Bispecific antigen binding proteins with fviii mimetic activity

Abstract

The present invention relates to FVIII mimicking antigen binding proteins. The present invention also relates to polynucleotides comprising a nucleotide sequence encoding a FVIII mimicking antigen binding protein, viral particles comprising the polynucleotides and the use of the antigen binding proteins and polynucleotides for therapy.

Description

Technical Field

[0001] This invention relates to antigen-binding proteins. It also relates to polynucleotides comprising a nucleotide sequence encoding an antigen-binding protein, viral particles comprising said polynucleotide, and treatments utilizing said antigen-binding protein and polynucleotide. Background Technology

[0002] Factor VIII (FVIII) is a key cofactor in the coagulation cascade, promoting the catalytic conversion of FX to activated FX (FXa) by forming a complex with activated factor IX (FIXa) and factor X (FX). In hemophilia A (an X-linked monogenic disorder with a prevalence of 1 in 5000 male births), quantitative and functional defects in FVIII disrupt the coagulation cascade, leading to life-threatening spontaneous bleeding symptom. As a standard treatment, FVIII replacement therapy requires frequent intravenous infusions of FVIII. However, approximately 30% of treated patients develop anti-FVIII neutralizing antibodies, leading to FVIII replacement therapy failure and significantly reduced life expectancy. Emicizumab, a bispecific antibody approved in late 2017, provides an alternative for patients with inhibitors through the development of associated FVIII mimic antibodies. In fact, FVIII mimic antibodies are a type of non-FVIII-based therapy. Their mechanism of action is to simultaneously bind to coagulation FIXa and FX, thereby promoting the catalytic conversion of FX into activated FXa under the action of FIXa, thus replacing one of the key functions of FVIII to maintain thrombin generation and blood clot formation.

[0003] The recent approval of gene therapy for hemophilia A (HA) using adeno-associated virus (AAV) marks a significant and long-awaited milestone in the development of therapeutic strategies for HA, as it promises a cure and overcomes the need for lifelong, periodic administration of factor concentrates or antibodies with FVIII mimicry activity, while significantly reducing bleeding events. However, the benefits of AAV gene therapy may be transient for patients with severe HA, as clinical data from recent clinical trials show that transgenic FVIII levels gradually decline over time. The reason for this decline in expression remains unexplained.

[0004] Therefore, improved treatment options are needed for HA, including gene therapy. Summary of the Invention

[0005] The inventors hypothesize that the gradual decline in transgenic FVIII levels observed over time in recent gene therapy clinical trials using AAV viral vectors is due to the use of oversized transgenic FVIII expression cassettes that reach the packaging limits of the viral capsids used to design these AAV vectors. Their hypothesis is supported by the fact that such a decline in transgenic expression has not been observed in other clinical gene therapy methods when the transgenic expression cassettes are within the packaging limits of the AAV capsid.

[0006] In view of the shortcomings of existing treatments, the inventors have developed a single-chain FVIII mimic antigen-binding protein (Bi8) for the treatment of diseases such as hemophilia A (HA). The antigen-binding protein of this invention exhibits FVIII mimic activity, successfully enhancing FIXa-mediated catalytic activation of FX, and demonstrating activity that fully corrects FVIII deficiency in hemophilia A plasma. The inventors then developed an improved gene therapy for HA that leverages the therapeutic potential of bispecific FVIII mimics using an expression cassette encoding a single-chain FVIII mimic antigen-binding protein, which remains within the packaging limitations of viral capsids used to design therapeutic AAV vectors. This approach provides an innovative solution for improving the prospect of long-term control of hemotropic factors in patients with severe HA.

[0007] This invention describes an innovative gene therapy strategy that utilizes an adeno-associated virus (AAV) vector to achieve stable transgenic expression of a coagulation factor VIII (FVIII) antigen-binding protein for the treatment of hemophilia A. This therapeutic approach relies on Bi8, a novel FVIII antigen-binding protein, designed as single-chain variable fragments (scFv) tandemly fused together to generate a single-chain bispecific antibody. This molecular form is smaller and less complex than conventional antibodies, making it particularly suitable for developing small-sized transgenic expression cassettes required for AAV-based vectors used in gene therapy.

[0008] This novel therapeutic strategy addresses existing limitations in the development of AAV-based gene therapies for hemophilia A, where the large size of the human coagulation FVIII coding sequence has led to the use of oversized transgene expression cassettes, while the regulatory elements are small. This may explain the progressive decline in transgene expression observed in patients participating in recent clinical trials. Furthermore, this non-FVIII-based strategy also provides a gene therapy solution for patients with FVIII-neutralizing antibodies who are receiving FVIII bypass agents but do not meet the eligibility criteria for existing hemophilia A gene therapy products.

[0009] A proof-of-concept was established in vitro to demonstrate the potential of FVIII mimics in the design of a single-chain bispecific antibody form of Bi8 and to confirm their ability to correct coagulation disorders in hemophilia A plasma. An AAV vector containing a liver-specific transgenic expression cassette with a Bi8 coding sequence was used to demonstrate stable transgenic expression of the FVIII mimic antibody and its therapeutic potential in a mouse model of hemophilia A.

[0010] Therefore, in a first aspect of the invention, an antigen-binding protein having FVIII mimicry activity is provided, which is composed of a single polypeptide chain.

[0011] The present invention also provides an antigen-binding protein composed of a single polypeptide chain, wherein the single polypeptide chain is composed of a first antigen-binding domain and a second binding domain, wherein the first antigen-binding domain selectively binds to coagulation FIX, and the second binding domain selectively binds to coagulation FX, and wherein each of the first antigen-binding domain and the second binding domain comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDR) 1, LCDR 2 and LCDR 3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR) 1, HCDR 2 and HCDR 3;

[0012] The first antigen-binding domain selectively binds to coagulation FIX; and

[0013] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0014] The present invention also provides a polynucleotide of no more than 5.0 kb in length, comprising a nucleic acid sequence encoding an antigen-binding protein as defined herein.

[0015] The present invention also provides a viral particle comprising polynucleotides as defined herein.

[0016] The present invention also provides a composition comprising an antigen-binding protein, a polynucleotide or viral particle as described herein, and a pharmaceutically acceptable excipient.

[0017] The present invention also provides an isolated host cell which has been transformed with polynucleotides or viral particles as defined herein.

[0018] The present invention also provides antigen-binding proteins, polynucleotides, viral particles, or compositions as defined herein for use in therapeutic applications.

[0019] The present invention also provides an AAV genome comprising a multinucleotide encoding an FVIII mimic antigen-binding protein for use in gene therapy. Attached Figure Description

[0020] Figure 1 Design of single-chain FVIII mimic antibodies

[0021] Bi8 was engineered as a single-chain FVIII mimic antibody, employing a bispecific tandem configuration of single-chain variable fragments (scFvs). Two scFvs targeting human coagulation FIX and FX are fused together via a flexible linker composed of glycine-serine repeat sequences. A 6x histidine motif is added to the C-terminus as a detection tag.

[0022] Figure 2 Size profile of recombinant Bi8 analyzed using SDS-Page analysis

[0023] Recombinant Bi8 antibody (recBi8) was produced in mammalian cells and purified by affinity chromatography. The resulting material was analyzed by Coomassie blue-stained SDS-Page gel under both reducing and non-reducing conditions, revealing a single separating band at 54.5 kDa.

[0024] Figure 3 FVIII mimicry activity of recBi8 in vitro

[0025] The FVIII mimic activity of recBi8 was evaluated in a dynamic colorimetric assay, which measures the antibody-enhanced conversion of FX to FXa via FIXa, using an equimolar concentration (40 nM) of the commercially available FVIII mimic antibody emicizumab as a baseline. Symbols represent the mean OD value ± SEM of n = 5 replicates. Control conditions were performed in the absence of the antibody.

[0026] Figure 4 The procoagulant potential of recBi8 in the thromboplastin time assay

[0027] The efficacy of recBi8 in correcting coagulation defects in human hemophilia A plasma was measured in vitro. 200 bethesda units (BU) of anti-FVIII antibody were added to a pool of citric acid-treated normal human plasma to neutralize endogenous FVIII. Then, 350 nM of recBi8 was added to this induced hemophilia A plasma, and the activated partial thromboplastin time (aPTT) was measured using a silica-based trigger. Symbols represent individual coagulation time values, and the horizontal line indicates the mean ± SD of three replicates. This indicates that the result is statistically significant (p < 0.0001) in both the one-way ANOVA test and the Sidak multiple comparisons.

[0028] Figure 5 Thrombin generation profile of recBi8 in hemophilia A plasma

[0029] The kinetics of thrombin formation were evaluated in a thrombin generation assay. 200 BU of anti-FVIII antibody was added to a citrated normal human plasma pool to neutralize endogenous FVIII, followed by the addition of 350 nM recBi8. Thrombin generation was then triggered using an FXIa-based trigger and measured using an automatically calibrated thrombin curve (CAT) method. (A) The curve represents the mean thrombin generation profile under various conditions within a 60-minute reading window. (B) The time to peak (time to maximum thrombin generation) and endogenous thrombin potential (ETP) were obtained from the thrombin generation plot and plotted as individual values ​​(symbols) and the mean of four independent replicates. ns indicates no statistical significance in a one-way ANOVA test versus Sidak multiple comparisons.

[0030] Figure 6 Design of a liver-specific expression cassette encoding Bi8 for AAV vectors

[0031] The cDNA sequence encoding Bi8 was cloned downstream of the liver control region enhancer and the human α-1 antitrypsin promoter (HCR-hAAT) to drive liver-specific strong expression of the transgene. (A) This expression cassette was further inserted between the inverted terminal repeat (ITR) sequences of AAV serotype 2 to generate a 4.4 kb single-stranded viral transgene construct. (B) An AAV vector encoding Bi8 (AAV_Bi8) was prepared using the AAV8 capsid and migrated on an alkaline gel to assess the integrity of the packaged transgene DNA. Controls were performed using AAV8 vectors prepared from transgene cassettes of 5.1 (high) and 4.3 (low) kb, respectively.

[0032] Figure 7 Transduction and expression of AAV_Bi8 in vitro

[0033] The expression and activity of Bi8 were evaluated after transduction of the human hepatocyte line HuH7 via AAV. An AAV vector encoding Bi8 (AAV_Bi8) was prepared using the AAV8 capsid serotype and expressed in a range of 10... 3 Up to 5x10 6 Huh7 cells were transduced at different multiples of infection (MOI) of vg / cell. (A) Transgenic Bi8 expression in cell culture supernatant was measured by ELISA at 48 hours post-infection. Symbols represent the mean ± SD of three replicates, and the dashed area indicates the lower limit of quantitation. (B) FVIII mimic activity of AAV-derived Bi8 was assessed by colorimetric assay of crude supernatant samples from the highest MOI, and the ratio of activity to antigen was normalized relative to recombinant Bi8 (recBi8). Symbols are individual values, and box-and-whisker plots represent the median and 25 / 75 quartile. ns indicates no statistical significance in nonparametric Mann-Whitney statistical analysis.

[0034] Figure 8 Characterization of AAV_Bi8 vector in a mouse model of hemophilia A

[0035] Using a mouse model of hemophilia A, we evaluated the AAV vector encoding the Bi8 sequence in vivo. The purified AAV vector was administered via a single intravenous injection into the lateral tail vein. Animals were monitored for up to 8 weeks to assess the expression and stability of the transgenic construct. The procoagulant effect of Bi8 after AAV-mediated gene transfer was further elicited in a tail vein transection hemorrhage model. AAV-treated mice were injected with a bolus dose of human FIX and FX, followed by tail vein transection 5 minutes later. Blood loss was recorded within 45 minutes post-transection.

[0036] Figure 9 Stable expression of Bi8 in AAV transduced animals

[0037] Mice with hemophilia A received a single intravenous injection of 4e 11 Or 4e 12 AAV_Bi8 vector was administered at vg / kg, with blood samples collected every 2 weeks. (A) The expression level of Bi8 in plasma samples following AAV vector infusion was then measured by ELISA. Symbols are mean ± SD for n=4 animals. (B) At 8 weeks post-infusion, the presence of anti-Bi8 antibodies (ADA) in the plasma of AAV-treated animals was further evaluated using a dedicated ELISA setup. Positive controls were performed by adding a known concentration of a 6x histidine-tagged monoclonal antibody against Bi8 to plasma samples. Symbols represent 3 replicates (positive control) or mean ± SD for individual animals. ND indicates not detected, and the dashed area represents the limit of quantitation in this assay.

[0038] Figure 10 Therapeutic efficacy of AAV_Bi8 vector in hemophilia A mice

[0039] Hemophilia A mice received a single intravenous injection of AAV_Bi8 vector at a dose of 4e. 11 4e 12 Or 1.2e 13 vg / kg. (A) Then, at 2 weeks post-AAV infusion, circulating plasma Bi8 levels were measured by ELISA. Symbols represent values ​​for individual animals, and box-and-whisker plots show the median and 25 / 75 quartile for each AAV dose. (B) Then, at 3 weeks post-AAV infusion, the bleeding phenotype of AAV-treated hemophilia A mice was validated in a tail vein transection model. Symbols represent blood loss per individual mouse, and box-and-whisker plots show the median and 25 / 75 quartile for each condition. Dashed lines show the median blood loss in untreated hemophilia A mice. and The p-values ​​indicate statistical significance (p < 0.01) in the one-way ANOVA test and the Sidak multiple comparison test, respectively.

[0040] Figure 11 The half-life of the single-chain antibody Bi8 is prolonged.

[0041] A Bi8 variant with an extended half-life (HLE) was engineered to confer albumin-binding capability, thereby benefiting from the FcRn recycling mechanism. A camel-derived, human albumin-targeting heavy-chain antibody fragment (VHH) was fused to the C-terminus of Bi8, downstream of the FX-targeting scFv, using a serine-glycine flexible linker. A 6x histidine motif for the detection antibody was added to the C-terminus of the albumin-binding VHH.

[0042] Figure 12 Characterization of recombinant Bi8-HLE

[0043] Recombinant antibody variants of Bi8 and Bi8-HLE were generated in mammalian cells and purified by affinity chromatography. (A) The resulting material was migrated onto a Coomassie blue-stained SDS-Page gel, and separate bands were observed at the expected size. (B) FVIII mimic activity was further evaluated in a dynamic colorimetric assay using incremental concentrations from 0.63 to 40 nM, and results are expressed as FXa activity. Symbols represent the mean of n = 3 replicates ± SD. Control conditions were performed in the absence of antibody.

[0044] Figure 13 AAV expression cassette encoding Bi8-HLE

[0045] (A) The cDNA sequence encoding Bi8-HLE was cloned into the same liver-specific expression cassette used for Bi8 and placed under the control of the (HCR-hAAT) promoter. The presence of albumin-binding VHH increased the total size of the cassette from 4.4 kb to 4.7 kb. (B) AAV vectors encoding Bi8 or Bi8-HLE were prepared using the AAV8 capsid serotype, and the integrity of the packaged transgenic DNA was assessed by alkaline gel electrophoresis.

[0046] Figure 14 Transducing Huh7 cells with an AAV vector encoding Bi8-HLE

[0047] The expression and activity of Bi8-HLE were evaluated after transduction of the human hepatocyte line HuH7 via AAV-mediated induction. The expression was assessed using an AAV8 vector encoding Bi8 or Bi8-HLE at a concentration of 1x10⁻⁶ cells / cells. 6 VG / cell was used to transduce cells, and antibody expression in the cell culture supernatant was measured by ELISA at 48 hours post-infection. Symbols represent individual values, and the bar chart is the mean ± SD of three replicates. ns indicates no statistical significance in the nonparametric Mann-Whitney statistical analysis. Invention Details

[0049] General definition

[0050] Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0051] Generally speaking, the term " Comprising "Intended to mean including but not limited to. For example, the phrase " Bag Polynucleotides containing nucleic acid sequences encoding the first antigen-binding domain "This should be interpreted as meaning that the polynucleotide has a nucleotide sequence that encodes the first antigen-binding domain, but the polynucleotide may also contain other nucleotides."

[0052] In some embodiments of the present invention, the word " Comprising Replace with phrase Group of... become ( consisting of )".the term" Composed of... ( consisting of The ) symbol is intended to be restrictive. For example, the phrase " A polynucleotide consisting of a nucleic acid sequence encoding a first antigen-binding domain "This should be understood as meaning that the polynucleotide has a nucleic acid sequence that encodes the first antigen-binding domain, but does not contain any other nucleotides."

[0053] the term" protein "and" polypeptide"These terms are used interchangeably in this article and are intended to refer to amino acid polymer chains of any length."

[0054] In this invention, to determine the percentage of identity between two sequences (such as two polynucleotide or two polypeptide sequences), these sequences are aligned to achieve optimal comparison (e.g., vacancies can be introduced in the first sequence to achieve optimal alignment with the second sequence). Subsequently, nucleotide residues at nucleotide positions are compared. When a position in the first sequence is occupied by the same nucleotide residue at the corresponding position in the second sequence, the nucleotides at that position are identical. The percentage of identity between two sequences is a function of the number of common positions shared by these sequences (i.e., identity % = number of common positions / total number of positions in the reference sequence x 100).

[0055] Typically, sequence comparisons are performed over the entire length of a reference sequence. For example, if a user wants to determine whether a given (“test”) sequence is 95% identical to SEQ ID NO. 19, then SEQ ID NO. 19 is the reference sequence. For instance, to assess whether a sequence is at least 80% identical to SEQ ID NO. 19 (an example of a reference sequence), a person skilled in the art would compare the entire length of SEQ ID NO. 19 and identify how many positions in the test sequence are identical to the corresponding positions in SEQ ID NO. 19. If at least 80% of the positions are identical, then the test sequence is at least 80% identical to SEQ ID NO. 19. If the sequence is shorter than SEQ ID NO. 19, empty or missing positions should be considered as dissimilar positions.

[0056] Those skilled in the art will recognize the various computer programs that can be used to determine homology or identity between two sequences. For example, mathematical algorithms can be used to perform sequence comparisons and determine the percentage of identity between two sequences. In one embodiment, the Needleman and Wunsch (1970) algorithm is used to determine the percentage of identity between two amino acid or nucleic acid sequences. This algorithm is integrated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), employing a Blosum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6, or 4 and length weights of 1, 2, 3, 4, 5, or 6.

[0057] For the purposes of this invention, the term " Excerpt"" refers to a continuous segment of a sequence. For example, the 50-amino acid segment in SEQ ID NO. 19 refers to the 50 consecutive amino acids in SEQ ID NO. 19.

[0058] antigen-binding proteins

[0059] An antigen can be defined as a compound, composition, or substance that can stimulate antibody production or elicit a T-cell response in an animal, including compositions injected or absorbed into an animal. Antigens react with products of specific humoral or cellular immunity, including products induced by heterologous antigens such as the disclosed antigens. An "epitaph" or "antigenic determinant" refers to a region of an antigen to which B cells and / or T cells respond. In one embodiment, T cells respond to an epitope when it is co-presented with an MHC molecule. Epitopes can be formed from consecutive amino acids or from the juxtaposition of non-consecutive amino acids by protein ternary folding. Epitopes formed from consecutive amino acids are generally retained upon exposure to denaturing solvents, while epitopes formed from ternary folding are generally lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3 amino acids, more commonly at least 5, about 9, or about 8-10 amino acids, exhibiting a unique spatial conformation. Methods for determining the spatial conformation of an epitope include, for example, X-ray diffraction crystallography and nuclear magnetic resonance.

[0060] Examples of antigens include, but are not limited to, peptides, lipids, polysaccharides, and nucleic acids containing antigenic determinants, such as those recognized by immune cells. Antigens may include peptides derived from pathogens of interest or from cancer cells. Exemplary pathogens include bacteria, fungi, viruses, and parasites. In some preferred embodiments, the antigen is FIX or FX or an antigenic fragment thereof.

[0061] A "target epitope" is a specific epitope on an antigen that specifically binds to an antibody of interest, such as a monoclonal antibody. In some examples, the target epitope includes an amino acid residue that contacts the antibody of interest, thus allowing selection of the target epitope by identifying the amino residue that contacts the antibody.

[0062] In a preferred embodiment of the present invention, the antigen-binding protein includes a first antigen-binding domain, wherein the first antigen-binding domain includes a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain includes light chain complementarity-determining regions (LCDR)1, LCDR2 and LCDR3, and wherein the heavy chain variable domain includes heavy chain complementarity-determining regions (HCDR)1, HCDR2 and HCDR3.

[0063] The antigen-binding proteins of the present invention include antibodies. The antigen-binding proteins of the present invention include single-chain antibodies (i.e., full-length heavy and light chains); Fab, modified Fab, Fab', modified Fab', F(ab')2, Fv, Fab-Fv, Fab-dsFv, single-domain antibodies (e.g., VH, VL, or VHH), scFv, monovalent antibodies, bivalent antibodies, trivalent or tetravalent antibodies, scFv, Bis-scFv, single-domain antibodies (sdAb) (also known as VHH antibodies), nanobodies (single-domain antibodies derived from camels), single-domain antibodies derived from shark IgNAR, diabody, tribody, triabody, tetrabody, and epitope-binding fragments of any of the above (see, for example, Holliger P and Hudson PJ, 2005, Nat. Biotechnol., 23: 1126-1136; Adair JR and Lawson ADG, 2005, DrugDesign Reviews – Online Edition, 2, 209-217). Methods for preparing and producing these antigen-binding proteins are well known in the art (see, for example, Verma R et al., 1998, J. Immunol. Methods, 216, 165-181). The Fab-Fv form was first disclosed in WO2009 / 040562, and its disulfide-stabilized form, Fab-dsFv, was first disclosed in WO2010 / 035012. The multivalent antigen-binding proteins of the present invention may contain multiple specificities, such as bispecificity, or may have monospecificity. The antigen-binding proteins of the present invention may be biparatopic.

[0064] scFv proteins are fusion proteins in which the variable regions of the light and heavy chains of immunoglobulins bind to each other via a linker. In dsFv, the chains are mutated to introduce disulfide bonds, thereby stabilizing the association between the two chains. The term also includes genetically engineered forms such as chimeric antibodies and heteroconjugate antibodies, such as bispecific antibodies. See also Pierce Catalog and Handbook , 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, Immunology , 3rd edition, WH Freeman & Co., New York, 1997.

[0065] The antigen-binding proteins of the present invention include, but are not limited to, the following: (1) Fab, a fragment containing a monovalent antigen-binding fragment of an antibody molecule, which is produced by digesting a whole antibody with papain to obtain a complete light chain and a portion of a heavy chain; (2) Fab', an antibody molecule fragment obtained by treating a whole antibody with pepsin and then reducing it to produce a complete light chain and a portion of a heavy chain; each antibody molecule yields two Fab' fragments; (3) (Fab')2, an antibody fragment obtained by treating a whole antibody with pepsin without subsequent reduction; (4) F(ab')2, a dimer consisting of two Fab' fragments held together by two disulfide bonds; (5) Fv, a genetically engineered fragment containing a light chain variable region and a heavy chain variable region, expressed as two chains; and (6) a single-chain antibody (“SCA”), a genetically engineered molecule containing a light chain variable region and a heavy chain variable region, which are linked by a suitable polypeptide linker to form a gene-fused single-chain molecule.

[0066] In one embodiment of the invention, the antigen-binding protein may comprise a heavy chain and a light chain, each chain containing a constant region and a variable region (these regions are also referred to as “domains”). In several embodiments of the invention, the heavy chain and light chain variable domains are combined to specifically bind the antigen. In other embodiments of the invention, only the heavy chain variable domain is required. For example, naturally occurring camel antibodies consist only of heavy chains and remain functional and stable in the absence of light chains (see, for example, Hamers-Casterman et al.). Nature , 363:446-448, 1993; Sheriff et al., Nat. Struct. Biol. , 3:733-736, 1996). The variable structural domains of light and heavy chains contain "framework" regions separated by three hypervariable regions, also known as "complementarity-determining regions" or "CDRs" (see, for example, Kabat et al., 3:733-736). Sequences of Proteins of Immunological Interest (US Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, which is a combined framework region of light and heavy chain components, functions to locate and arrange the CDRs in three-dimensional space.

[0067] The frame region (CDR) is primarily responsible for antigen binding. The sequences of the frame regions of different light or heavy chains are relatively conserved within a species. The frame region of an antibody, which is a combined frame region of light and heavy chain components, functions to locate and arrange the CDR in three-dimensional space.

[0068] Each chain's CDR is typically referred to as CDR1, CDR2, and CDR3 (from the N-end to the C-end), and is usually identified by the chain to which the specific CDR belongs. Therefore, V H CDR3 is located in the variable domain of its host antibody heavy chain, while V L CDR1 is derived from the variable domain of the antibody light chain. Light chain CDRs can also be called CDR L1, CDR L2, and CDR L3, or LCDR1, LCDR2, and LCDR3. Heavy chain CDRs can be called CDR H1, CDR H2, and CDR H3, or HCDR1, HCDR2, and HCDR3.

[0069] Residues in the variable domain of an antibody are typically numbered according to IMGT (http: / / www.imgt.org). This system is described in Lefranc MP (1997, J, Immunol. Today, 18, 509). Unless otherwise stated, this numbering system is used throughout this specification.

[0070] The designation of IMGT residues does not always correspond directly to the linear numbering of amino acid residues. The actual linear amino acid sequence may contain fewer or more amino acids than a strict IMGT number, corresponding to a shortening or insertion of a structural component in a basic variable domain structure, whether that component is a framework or a CDR. For a given antibody, the correct IMGT number of its residues can be determined by comparing homologous residues in the antibody sequence with the sequence of “standard” IMGT numbers.

[0071] Suitable antigen-binding proteins, antibodies, or their binding fragments are disclosed herein by means of their heavy and light chain CDRs, their heavy and light chain variable regions, and / or their full-length heavy and light chain primary amino acid sequences.

[0072] An antigen-binding protein, antibody, or binding fragment thereof may comprise one or more VH CDR sequences of the specific antigen-binding protein or antibody, and alternatively or additionally, may comprise one or more VL CDR sequences in addition to VL CDR1. An antigen-binding protein, antibody, or binding fragment thereof may comprise one, two, or all three VH CDR sequences of the specific antigen-binding protein, antibody, or binding fragment thereof as described above, and alternatively or additionally, may comprise one, two, or all three VL chain CDR sequences of the specific antigen-binding protein, antibody, or binding fragment thereof, including VL CDR1. An antigen-binding protein, antibody, or binding fragment thereof may comprise all six CDR sequences of the specific antigen-binding protein, antibody, or binding fragment as described above.

[0073] Variant antigen-binding proteins or antibodies may contain 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions relative to specific sequences and fragments discussed above, while still retaining the activity of the antigen-binding proteins or antibodies described herein. "Deletion" variants may include the deletion of, for example, 1, 2, 3, 4 or 5 amino acids individually, or the deletion of one or more groups of amino acids, such as groups of 2, 3, 4 or 5 amino acids. An "amino acid group" can be defined as one that is consecutive to each other, or one that is very close to each other but not consecutive. "Substitution" variants preferably involve replacing one or more amino acids with the same number of amino acids, and are conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, another aliphatic amino acid, another small amino acid, another small amino acid, or another large amino acid. Some properties of the 20 major amino acids can be used to select suitable substitutes, as shown below:

[0074]

[0075] Preferred "derivatives" or "variants" include cases where the amino acids appearing in the sequence are not naturally occurring amino acids, but rather structural analogs of them. The amino acids used in the sequence may also be derivatized or modified, such as labeled, provided that the function of the antibody is not significantly adversely affected.

[0076] The derivatives and variants described above can be prepared during the synthesis of antigen-binding proteins or antibodies, or through post-production modification, or, when the antigen-binding proteins or antibodies are in recombinant form, using known site-directed mutagenesis, random mutagenesis, or enzymatic cleavage and / or ligation techniques.

[0077] Preferably, the amino acid sequence of the variant antigen-binding protein or antibody has greater than 60% or greater than 70%, such as 75% or 80%, amino acid identity with the VL and / or VH or fragments thereof of the antigen-binding protein or antibody disclosed herein, preferably greater than 85%, such as greater than 90%, 95%, 96%, 97%, 98%, or 99% amino acid identity. This level of amino acid identity may occur throughout the full length of the relevant SEQ ID NO sequence, or within a portion of that sequence, such as spanning 20, 30, 50, 75, 100, 150, 200, or more amino acids, depending on the size of the full-length polypeptide.

[0078] Preferably, the variant antigen-binding protein or antibody comprises one or more CDR sequences as described herein.

[0079] In terms of amino acid sequence, "sequence identity" refers to a sequence that has a specified value when evaluated using ClustalW (Thompson JD et al., 1994, Nucleic Acid Res., 22, 4673-4680), with the following parameters:

[0080] Pairing parameters - method: slow / precise, matrix: PAM, void opening penalty: 10.00, void extension penalty: 0.10;

[0081] Multiple sequence alignment parameters - matrix: PAM, vacancy opening penalty: 10.00, delayed identity %: 30, penalized terminal vacancy: on, vacancy spacing: 0, negative matrix: no, vacancy extension penalty: 0.20, residue-specific vacancy penalty: on, hydrophilic vacancy penalty: on, hydrophilic residues: G, P, S, N, D, Q, E, K, R. Sequence identity at a specific residue is intended to include identical residues that have only undergone derivatization.

[0082] The method of the present invention can use antibodies having specific VH and VL amino acid sequences, as well as their variants and fragments, which maintain the function or activity of these VH and VL.

[0083] Mentioning "V" H "VH" or "VH" refers to the variable region of the immunoglobulin heavy chain, including the variable region of antibody fragments such as Fv, scFv, dsFv, or Fab. Mentioning "V"... L "VL" or "VL" refers to the variable region of the immunoglobulin light chain, including the variable region of the light chain of Fv, scFv, dsFv, or Fab.

[0084] In some embodiments of the invention, the antigen-binding protein may be an antibody comprising heavy (H) chains and light (L) chains linked together by disulfide bonds. There are two types of light chains: lambda (λ) and kappa (κ). The heavy chains are primarily of five classes (or isotypes) that determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0085] IgG1 antibodies (e.g., IgG1 / κ) having both a heavy and light chain are advantageously used in this invention. However, this invention also covers other human antibody isotypes, including IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, and IgE combined with a κ or λ type light chain. Furthermore, this invention can use antibodies of all animal origins of various isotypes. Antibodies can be full-length antibodies or antigen-binding fragments of antibodies, including Fab, F(ab')2, single-chain Fv fragments, or single-domain VHH, VH, or VL domains.

[0086] The Fc region typically refers to the last two constant immunoglobulin domains of IgA, IgD, and IgG, and the last three constant immunoglobulin domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge located at the N-terminus of these domains. For IgA and IgM, the Fc region may or may not contain a tailpiece and may or may not bind to the J chain. For IgG, the Fc region contains the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3), and the lower portion of the hinge located between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of the Fc region may vary, the Fc region of the human IgG heavy chain is generally defined as the region encompassing residue C226 or P230 to its carboxyl terminus, numbered according to the EU index. For IgA, the Fc region comprises the immunoglobulin domains Calpha2 and Calpha3 (Cα2 and Cα3), and the lower part of the hinge located between Calpha1 (Cα1) and Cα2. The definition of the Fc region encompasses functionally equivalent analogs and variants of the Fc region. Functionally equivalent analogs of the Fc region may be variant Fc regions that contain one or more amino acid modifications relative to the wild-type or naturally occurring Fc region. Variant Fc regions will have at least 50% homology with the naturally occurring Fc region, such as about 80% and about 90% or at least about 95% homology. Functionally equivalent analogs of the Fc region may contain one or more amino acid residues added to or deleted from the N-terminus or C-terminus of the protein, such as additions and / or deletions of no more than 30 or no more than 10. Functionally equivalent analogs of the Fc region include Fc regions operatively linked to a fusion chaperone. Functionally equivalent analogues to the Fc region must contain a majority of all Ig domains constituting the Fc region as defined above; for example, the IgG and IgA Fc regions as defined herein must contain a majority of the sequence encoding CH2 and a majority of the sequence encoding CH3. Therefore, a single CH2 domain or a single CH3 domain is not considered an Fc region. The Fc region can refer to this region in its isolated state or within the context of an Fc fusion peptide.

[0087] When referring to the antigen-binding protein of the present invention, the binding of the protein to the antigen refers to a binding reaction that identifies the presence of a target protein, peptide, or polysaccharide in the presence of a heterogeneous population of proteins and other biological products. Therefore, under specified conditions, the antigen-binding protein preferentially binds to a specific target protein, peptide, or polysaccharide (such as FIX or FX) without binding in significant amounts to other proteins or polysaccharides present in the sample or subject. Specific binding can be determined by methods known in the art. With respect to antibody-antigen complexes, the Kd for specific binding between the antigen and antibody is less than about 10. -5 M, 10 -6 M, 10 -7 M, such as less than about 10 -7 M, 10 -8 M, 10 -9 M, or even less than about 10 -10 M.

[0088] The terms "binding activity" and "binding affinity" refer to the tendency of an antigen-binding protein to bind to or not bind to a target. Binding affinity can be quantified by measuring the dissociation constant (Kd) between the antigen-binding protein and its target. Similarly, the specificity of an antigen-binding protein binding to its target can be defined by the relative dissociation constant (Kd) of the antibody relative to its target, which is relative to the dissociation constant between the antigen-binding protein and another non-target molecule.

[0089] Typically, the Kd of an antibody against its target will be 2 times lower, preferably 5 times lower, and more preferably 10 times lower than its Kd against other non-target molecules such as irrelevant substances or accompanying substances in the environment. More preferably, the Kd will be 50 times lower, even more preferably 100 times lower, and still more preferably 200 times lower.

[0090] The value of this dissociation constant can be determined directly by well-known methods, and even for complex mixtures, it can be calculated by methods such as those described in Caceci MS and Cacheris WP (1984, Byte, 9, 340-362). For example, Kd can be determined using a double-filtration nitrocellulose membrane combined with a determination method, such as the one disclosed by Wong I and Lohman TM (1993, Proc. Natl. Acad. Sci. USA, 90, 5428-5432), or, for example, by using Octet surface plasmon resonance.

[0091] One method for assessing binding affinity is ELISA. Other standard assays for assessing the binding ability of ligands such as antibodies to targets are also known in the art, including, for example, Western blotting, RIA, and flow cytometry. Antibody binding kinetics (e.g., binding affinity) can also be assessed using standard assays known in the art, such as surface plasmon resonance, for example, via the Biacore™ system analysis.

[0092] In one embodiment, the antigen-binding protein is a monoclonal antibody. Monoclonal antibodies are identical immunoglobulin molecules with a single binding specificity and affinity for a specific epitope. The monoclonal antibodies (mAbs) of the present invention can be produced by a variety of techniques, including conventional monoclonal antibody methodologies, such as those disclosed in "Monoclonal Antibodies: a manual of techniques" (Zola H, 1987, CRC Press) and "Monoclonal Hybridoma Antibodies: techniques and applications" (Hurrell JGR, 1982, CRC Press).

[0093] In a preferred embodiment of the present invention, the antigen-binding protein comprises a first antigen-binding domain and a second antigen-binding domain. In a preferred embodiment of the present invention, the first and second antigen-binding domains of the antigen-binding protein are scFv proteins. In a preferred embodiment of the present invention, the first and second antigen-binding domains of the antigen-binding protein are scFv proteins covalently linked via peptide linkers.

[0094] As shown below, the sequences of the light chain variable domains and heavy chain variable domains mentioned above may differ from the given sequences. For example, a light chain / heavy chain variable domain may contain a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence listed in the sequence listing. Alternatively, the light chain / heavy chain variable domain sequences may differ at up to 10 amino acid positions, but preferably the number of amino acid substitutions is less than 10, and therefore may have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution.

[0095] As mentioned above, in some embodiments, the light chain variable domain and heavy chain variable domain comprise amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown above. For example, the light chain framework region, heavy chain framework region, light chain variable domain, and heavy chain variable domain may include up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in the amino acid sequences shown above. When variations exist in the sequences of the light chain variable domain and heavy chain variable domain, any amino acid substitution is preferably not located in the CDR. Specifically, the light chain framework region and / or heavy chain framework region of the above-described antibody may comprise amino acid sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequences shown above. Furthermore, the light chain framework region and / or heavy chain framework region may include up to 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions in the amino acid sequence shown above. Preferably, the amino acid substitutions are conserved substitutions as described above. For example, the framework region may contain such substitutions to achieve sequence humanization. Preferably, the framework region is humanized.

[0096] The sequences of each light chain variable domain and heavy chain variable domain of the second antigen-binding domain mentioned above may differ from the given sequence. For example, the light chain / heavy chain variable domain may contain a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence listed in the sequence listing. Alternatively, the light chain / heavy chain variable domain sequences may differ at up to 10 amino acid positions, but preferably the number of amino acid substitutions present is less than 10, and therefore may have up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitution. Preferably, there are no substitutions in the CDR of the heavy chain / light chain.

[0097] Antigen-binding proteins containing antigen-binding domains can be prepared by proteolytic hydrolysis of antibodies or by expressing DNA encoding the fragment in mammalian cells. Antibody fragments can be obtained by conventional methods, such as digestion of whole antibodies with pepsin or papain. For example, antibody fragments can be generated by enzymatic cleavage of antibodies with pepsin, providing a 5S fragment called F(ab')2. This fragment can be further cleaved with a thiol reducing agent, optionally with a blocking group targeting the thiol group generated by the cleavage of disulfide bonds, to generate a 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage with pepsin directly generates two monovalent Fab' fragments and an Fc fragment (see U.S. Patent Nos. 4,036,945 and 4,331,647, and the references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology Volume 1, page 422, Academic Press, 1967; and Coligan et al., see sections 2.8.1–2.8.10 and 2.10.1–2.10.4).

[0098] Other methods can also be used to cleave antigen-binding proteins and antibodies, such as separating the heavy chain to form a monovalent light-heavy chain fragment, further cleaving the fragment, or using other enzymatic, chemical, or genetic techniques, as long as the fragment can bind to an antigen recognized by the intact antibody or antigen-binding protein.

[0099] In a preferred embodiment, the antigen-binding protein has FVIII-mimicking activity and is composed of a single polypeptide chain.

[0100] In a preferred embodiment, the single polypeptide chain is composed of a first antigen-binding domain and a second antigen-binding domain.

[0101] In a preferred embodiment, the single polypeptide chain is composed of a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain selectively binds to coagulation FIX and the second antigen-binding domain selectively binds to coagulation FX.

[0102] In a preferred embodiment, the antigen-binding protein comprises a first antigen-binding domain that selectively binds to FIX / FIXa and a second antigen-binding domain that selectively binds to FX / FXa.

[0103] In a preferred embodiment, the first antigen-binding domain and the second antigen-binding domain each include a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain includes light chain complementarity-determining regions (LCDRs) 1, LCDR 2, and LCDR 3, and wherein the heavy chain variable domain includes heavy chain complementarity-determining regions (HCDRs) 1, HCDR 2, and HCDR 3.

[0104] The first antigen-binding domain selectively binds to coagulation FIX; and

[0105] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12;

[0106] The sequence of each complementarity-determining region can differ from the given sequence at up to two amino acid positions.

[0107] In a preferred embodiment, the first antigen-binding domain and the second antigen-binding domain each include a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain includes light chain complementarity-determining regions (LCDRs) 1, LCDR 2, and LCDR 3, and wherein the heavy chain variable domain includes heavy chain complementarity-determining regions (HCDRs) 1, HCDR 2, and HCDR 3.

[0108] For the first antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 1; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 2; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 3; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 4; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 5; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6; and

[0109] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12;

[0110] The sequence of each complementarity-determining region can differ from the given sequence at up to two amino acid positions.

[0111] In a specific embodiment, for the first antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13, and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14; and for the second antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 16.

[0112] In a particular embodiment, a first antigen-binding domain is contained in the scFv. In a particular embodiment, a second antigen-binding domain is contained in the scFv.

[0113] In a particular embodiment, the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 17. In a particular embodiment, the second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 18.

[0114] In a particular embodiment, the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 17, and the second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 18. In a particular embodiment, the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO: 19. The Bi8 antibody mentioned herein comprises the amino acid sequence shown in SEQ ID NO: 19.

[0115] Antigen-binding proteins using single-stranded tandem scFvs typically have a short half-life (1-2 hours) in humans. However, partially fusing an scFv-based antigen-binding protein with a protein that interacts with the neonatal Fc receptor (FcRn) pathway can extend the circulating half-life through an FcRn-mediated recycling mechanism. Therefore, in embodiments using viral vectors such as AAV for gene transfer administration of antigen-binding proteins, expressing an antigen-binding protein with a longer half-life will increase its steady-state levels, potentially reducing the vector dose required to achieve therapeutic efficacy.

[0116] Therefore, in one embodiment of the present invention, the half-life of the antigen-binding protein is extended.

[0117] In a preferred embodiment, the antigen-binding protein further comprises a third antigen-binding domain that binds to proteins that interact with FcRn.

[0118] Those skilled in the art will understand that alternative third antigen-binding domains that directly interact with FcRn can be selected.

[0119] In a specific implementation, the third antigen-binding domain binds to albumin.

[0120] In a specific embodiment, the third antigen-binding domain comprises CDR1, CDR2, and CDR3 sequences, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 42; CDR2 comprises the amino acid sequence shown in SEQ ID NO: 43; and CDR3 comprises the amino acid sequence shown in SEQ ID NO: 44.

[0121] In a preferred embodiment, the third antigen-binding domain comprises a VHH domain.

[0122] In a preferred embodiment, the VHH domain is derived from camels.

[0123] Those skilled in the art will understand that suitable alternative truncated forms of the third antigen-binding domain that can be used include VHH antibodies, recombinant antibodies, single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen-binding regions (Fab), single-domain antibodies (sdAb), nanobodies, single-domain antibodies derived from camels, single-domain antibody fragments derived from shark IgNAR (VNAR), bisomal antibodies, three-armed antibodies, anticarrier proteins, and aptamers.

[0124] In a preferred embodiment, the third antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 41. SEQ ID NO: 41 corresponds to the amino acid sequence of the albumin-targeting VHH. This albumin-targeting VHH is also referred to herein as "…". HLE ".

[0125] In a preferred embodiment, the extended half-life antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO: 40.

[0126] Polynucleotides

[0127] This invention provides polynucleotides of length not exceeding 4.5 kb, 4.6 kb, 4.7 kb, 4.8 kb, 4.9 kb, or 5.0 kb, encoding FVIII mimic antigen-binding protein sequences as defined herein. This invention also provides polynucleotides of length not exceeding 5.0 kb, encoding FVIII mimic antigen-binding protein sequences as defined herein.

[0128] The terms "sequence encoding..." or "polynucleotide encoding..." refer to a nucleotide sequence containing a codon encoding the encoded polypeptide. For example, a nucleotide sequence encoding an antigen-binding protein or a fragment thereof contains a codon encoding the amino acid sequence of that antigen-binding protein or fragment thereof. A suitable nucleotide sequence of the present invention is provided in SEQ ID NO. 21.

[0129] The following table describes the codons that encode each amino acid:

[0130]

[0131] The corresponding RNA codons will contain U instead of T in the table above.

[0132] One aspect of the present invention provides a polynucleotide comprising an antigen-binding protein nucleotide sequence, wherein the antigen-binding protein nucleotide sequence encodes an antigen-binding protein or a fragment thereof, and has at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with SEQ ID NO. 21.

[0133] Generally, the antigen-binding protein nucleotide sequence can have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with a fragment of at least 1200, at least 1350, or at least 1530 nucleotides of SEQ ID NO. 21, and retain FVIII mimicry activity. The antigen-binding protein nucleotide sequence can have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with a continuous fragment of at least 1200, at least 1350, or at least 1530 nucleotides of SEQ ID NO. 21, and retain FVIII mimicry activity. The antigen-binding protein nucleotide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with SEQ ID NO. 21, and retain FVIII mimicry activity. For example, the antigen-binding protein nucleotide sequence may have at least 98% identity with SEQ ID NO. 21 and retain FVIII mimicry activity.

[0134] In one embodiment of the invention, the antigen-binding protein nucleotide sequence may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identity with SEQ ID NO 21 and retain FVIII mimicry activity, wherein the CDR nucleotide sequences of the first and second antigen-binding domains encode the amino acid sequences shown in SEQ ID NO 1 to 12.

[0135] In a preferred embodiment of the polynucleotide sequence of the present invention, the antigen-binding protein nucleotide sequence encodes an antigen-binding protein, wherein the antigen-binding protein optionally further comprises a third antigen-binding domain, wherein the third binding domain binds to a protein that interacts with FcRn, and wherein the length of the polynucleotide sequence does not exceed 5.0 kb, thereby enabling the polynucleotide sequence to be packaged (or adapted) in a single AAV vector.

[0136] In a preferred embodiment, the antigen-binding protein further comprises a third antigen-binding domain that binds to proteins that interact with FcRn.

[0137] Those skilled in the art will understand that alternative third antigen-binding domains that directly interact with FcRn can be selected.

[0138] In a specific implementation, the third antigen-binding domain binds to albumin.

[0139] In a specific embodiment, the nucleic acid sequence encodes a third antigen-binding domain comprising CDR1, CDR2, and CDR3 sequences, wherein the nucleic acid sequence encoding CDR1 is shown in SEQ ID NO: 47; the nucleic acid sequence encoding CDR2 is shown in SEQ ID NO: 48; and the nucleic acid sequence encoding CDR3 is shown in SEQ ID NO: 49.

[0140] In a preferred embodiment, the third antigen-binding domain comprises a VHH domain.

[0141] In a preferred embodiment, the VHH domain is derived from camels.

[0142] Those skilled in the art will understand that suitable alternative truncation forms targeting the third binding domain can be selected, resulting in a multinucleotide sequence length equal to or less than 5.0 kb. Therefore, those skilled in the art will select suitable forms targeting the third antigen-binding domain with a length equal to or less than 5.0 kb, such as VHH antibodies, recombinant antibodies, single-chain antibodies, single-chain variable fragments (scFv), variable fragments (Fv), fragment antigen-binding regions (Fab), single-domain antibodies (sdAb), nanobodies, single-domain antibodies derived from camels, single-domain antibody fragments derived from shark IgNAR (VNAR), bisomal antibodies, three-armed antibodies, anticarrier proteins, aptamers, and peptides.

[0143] Those skilled in the art will understand that a suitable form targeting the third antigen-binding domain may include three CDR sequences, namely CDR1, CDR2 and CDR3.

[0144] In a preferred embodiment, the nucleic acid sequence encoding the third antigen-binding domain comprises the nucleic acid sequence shown in SEQ ID NO:46. SEQ ID NO:46 corresponds to the nucleotide sequence of a VHH targeting albumin. This VHH targeting albumin is also referred to herein as "…". HLE ".

[0145] Therefore, a suitable polynucleotide sequence of the present invention comprises SEQ ID NO: 45, which corresponds to the nucleotide sequence of the antigen-binding protein of the present invention comprising the third antigen-binding domain as described herein.

[0146] Polynucleotides may further contain promoter / transcriptional regulatory elements.

[0147] Polynucleotides can contain promoters. These promoters contain transcriptional regulatory elements. Any suitable transcriptional regulatory element can be used, such as HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and LSP, all of which are liver-specific transcriptional regulatory elements. For a more detailed description of these transcriptional regulatory elements, please see the following references: HLP1: McIntosh J. et al., Blood, April 25, 2013, 121(17):3335-44; LP1: Nathwani et al., Blood, April 1, 2006, 107(7): 2653–2661; HCR-hAAT: Miao et al. , MolTher. 2000;1: 522-532; ApoE-hAAT: Okuyama et al. , Human Gene Therapy, 7, 637-645 (1996); and LSP: Wang et al. , Proc Natl Acad Sci US A. March 30, 1999, 96(7): 3906–3910.

[0148] Transcriptional regulatory elements may include promoters and / or enhancers, such as promoter and / or enhancer elements from HLP2, HLP1, LP1, HCR-hAAT, ApoE-hAAT, and LSP. Each of these transcriptional regulatory elements contains a promoter, an enhancer, and optional other nucleotides.

[0149] In one embodiment, the transcriptional regulatory element comprises an enhancer that is the human apolipoprotein E (ApoE) liver locus control region (HCR; Miao et al. (2000), Molecular Therapy 1(6):522) or a fragment thereof. In one embodiment, the transcriptional regulatory element comprises a fragment of the HCR enhancer having a length of at least 80, at least 90, at least 100, less than 192, 80 to 192, 90 to 192, 100 to 250, or 117 to 192 nucleotides. Optionally, the HCR enhancer fragment has a length of 100 to 250 nucleotides.

[0150] If the polynucleotide is intended to be expressed in the liver, the promoter can be a liver-biased or liver-specific promoter. Optionally, the promoter is a human liver-specific promoter.

[0151] “ liver-preferred promoters"This is a promoter primarily expressed in the liver and related tissues. For example, those skilled in the art can determine whether a promoter is a liver-preferred promoter by comparing the expression of the polynucleotide in hepatocytes (such as Huh 7 cells) with the expression of the same polynucleotide in cells from other tissues." Liver-specific Sex promoters "Liver-specific" refers to a promoter that typically provides a higher expression level in hepatocytes compared to other cells. For example, those skilled in the art can determine whether a promoter is liver-specific by comparing the expression of a polynucleotide in hepatocytes (such as Huh 7 cells) with the expression of that polynucleotide in cells from other tissues. If the expression level in hepatocytes is higher compared to cells from other tissues, then the promoter is liver-specific.

[0152] In a preferred embodiment, the promoter may comprise a liver control region enhancer / human α-1 antitrypsin promoter (HCR hAAT) complex.

[0153] In a preferred embodiment, the promoter may contain CAG introns.

[0154] In a preferred embodiment, the promoter may comprise a liver control region enhancer / human α-1 antitrypsin promoter (HCR hAAT) complex and a CAG intron.

[0155] Viral particles containing polynucleotides

[0156] The present invention further provides viral particles comprising the polynucleotides of the present invention. For the purposes of this invention, the term "viral particle" refers to all or part of a virion. For example, a viral particle comprises a recombinant genome and may further comprise a capsid. A viral particle can be a gene therapy vector. In this document, the term "viral particle"... Virus particles "and" carrier "They can be used interchangeably. For the purposes of this application," Gene therapy "A vector is a viral particle that can be used in gene therapy. It is a viral particle that contains all the functional elements required to express a transgene (such as the FVIII mimic antigen-binding protein) in a host cell after administration."

[0157] Suitable viral particles include parvovirus, retrovirus, lentivirus, or herpes simplex virus. Parvovirus may be adeno-associated virus (AAV). The viral particles are preferably recombinant adeno-associated virus (AAV) vectors or lentiviral vectors. More preferably, the viral particles are AAV viral particles. The terms AAV and rAAV are used interchangeably herein.

[0158] All known AAV serotypes share a very similar genomic structure. The AAV genome is a linear single-stranded DNA molecule less than approximately 5,000 nucleotides in length. Inverted terminal repeats (ITRs) are located flanking the unique coding nucleotide sequences of non-structural replication (Rep) and structural (VP) proteins. VP proteins (VP1, VP2, and VP3) form the capsid. The terminal 145 nt is self-complementary and organized in a way that allows for the formation of an energy-stable intramolecular double helix, resulting in T-shaped hairpins. These hairpin structures serve as the starting point for viral DNA replication and act as primers for the cellular DNA polymerase complex. After mammalian cells are infected with wild-type (wt) AAV, the Rep genes (encoding Rep78 and Rep52 proteins) are expressed under the action of the P5 and P19 promoters, respectively, and both Rep proteins play a role in viral genome replication. Splicing events occur in the Rep ORF, resulting in the actual expression of four Rep proteins (Rep78, Rep68, Rep52, and Rep40). However, it has been shown that in mammalian cells, unspliced ​​mRNAs encoding Rep78 and Rep52 proteins are sufficient for the production of AAV vectors. Similarly, in insect cells, Rep78 and Rep52 proteins are also sufficient for the production of AAV vectors.

[0159] The recombinant viral genome of the present invention may contain an ITR. The AAV vector of the present invention may function using only one ITR. Therefore, the viral genome contains at least one ITR, but more typically contains two ITRs (usually one at each end of the viral genome, i.e., one at the 5' end and the other at the 3' end). Intercalation sequences may exist between the polynucleotide and one or more ITRs. The polynucleotide of the present invention can be integrated into the viral particle, positioned between two conventional ITRs, or positioned on either side of an ITR engineered using two D regions.

[0160] The AAV sequences used in this invention to generate AAV vectors can be derived from the genome of any AAV serotype. Generally, AAV serotypes have highly homologous genome sequences at the amino acid and nucleic acid levels, providing the same set of genetic functions, producing virions that are substantially identical in physical and functional form, and replicating and assembling through almost identical mechanisms. For an overview of the genome sequences and genome similarities of various AAV serotypes, see, for example, GenBank accession numbers U89790; J01901; AF043303; AF085716; Chiorini et al., 1997; Srivastava et al., 1983; Chiorini et al., 1999; Rutledge et al., 1998; and Wu et al., 2000. This invention can use AAV serotypes 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9, 10, 11, or 12. When used to prepare gene therapy vectors, sequences from AAV serotypes can be mutated or engineered.

[0161] Optionally, the AAV vector contains an ITR sequence derived from AAV1, AAV2, AAV4, and / or AAV6. Preferably, the ITR sequence is an AAV2 ITR sequence. In this document, the term AAVx / y refers to a viral particle containing components from AAVx (where x is an AAV serotype number) and components from AAVy (where y is a number of the same or different serotype). For example, an AAV2 / 8 vector may contain a portion of the viral genome from an AAV2 strain, including the ITR, and a capsid derived from an AAV8 strain.

[0162] The virus particles of this invention can be a type of " hybrid "Particles in which the viral ITR and viral capsid originate from different parvoviruses, such as different AAV serotypes. Preferably, the viral ITR and capsid originate from different serotypes of AAV; in this case, such viral particles are referred to as transcapitalized particles or pseudotyped particles. Similarly, parvoviruses can have..." Inlay "Capsid (e.g., containing sequences from different parvoviruses, preferably different AAV serotypes) or " Targeted "Capsule (e.g., directional craving)."

[0163] In some embodiments, the recombinant AAV genome contains a complete ITR containing a functional end-resolved site (TRS). This AAV genome may contain one or two resolveable ITRs, i.e., ITRs containing functional TRSs, where site-specific cleavage can occur to produce a free 3' hydroxyl group, which can serve as a substrate for DNA polymerase to unwind and replicate the ITR. Preferably, the recombinant genome is single-stranded (i.e., it is packaged in the viral particle in single-stranded form). Optionally, the recombinant genome is not packaged in a self-complementary conformation, i.e., the genome does not contain a single covalently linked polynucleotide chain having a substantially self-complementary portion annealed in the viral particle. Alternatively, the recombinant genome may be packaged in a… Monomer double chain Packaging is done in the form of "". This is described in WO 2011 / 122950. Monomer double chain The genome can be packaged as two substantially complementary but non-covalently linked polynucleotides and annealed within the viral particle.

[0164] Viral particles may further contain poly-A sequences or polyadenylated sequences. The poly-A sequence may be downstream of the nucleotide sequence encoding the antigen-binding protein. The poly-A sequence may be a bovine growth hormone poly-A sequence (bGHpA). The length of the poly-A sequence may be 250 to 270 nucleotides. The poly-A sequence may be downstream of the nucleotide sequence encoding the antigen-binding protein and upstream of the ITR.

[0165] This invention covers a viral particle comprising a polynucleotide of no more than 5.0 kb in length, the polynucleotide encoding a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDRs) 1, LCDR 2, and LCDR 3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR 2, and HCDR 3;

[0166] For the first antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 1; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 2; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 3; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 4; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 5; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, and...

[0167] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0168] The present invention includes an AAV genome containing a multinucleotide encoding an FVIII mimic antigen-binding protein, which is used in gene therapy.

[0169] host cells

[0170] This invention covers isolated host cells transformed with polynucleotides or viral particles as defined herein. Suitable host cells include cultured human hepatocytes, such as Huh 7 cells. Suitable host cells for producing antigen-binding proteins or antibodies include mammalian cells or *Escherichia coli* (E. coli). E. coli ).

[0171] Compositions, methods and uses

[0172] In another aspect of the invention, a composition is provided comprising the antigen-binding protein of the invention, a polynucleotide or a carrier / virus particle, and a pharmaceutically acceptable excipient.

[0173] Pharmaceutically acceptable excipients may include carriers, diluents, and / or other pharmaceutical preparations, drug preparations, or adjuvants. Optionally, pharmaceutically acceptable excipients include saline solutions. Optionally, pharmaceutically acceptable excipients include human serum albumin.

[0174] Preferably, the carrier is suitable for parenteral administration, such as intravenous, intraocular, intramuscular, subcutaneous, intradermal, or intraperitoneal administration (e.g., by injection or infusion). In some embodiments, pharmaceutically acceptable carriers include at least one carrier selected from the group consisting of: cosolvent solutions, liposomes, micelles, liquid crystals, nanocrystals, nanoparticles, emulsions, microparticles, microspheres, nanospheres, nanocapsules, polymers or polymeric carriers, surfactants, suspending agents, complexing agents such as cyclodextrins or adsorbent molecules such as albumin, surfactant particles, and chelating agents. In another embodiment, the polysaccharide includes hyaluronic acid and its derivatives, dextran and its derivatives, cellulose and its derivatives (e.g., methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, cellulose acetate phthalate, cellulose acetate succinate, cellulose acetate butyrate, hydroxypropylmethylcellulose phthalate), chitosan and its derivatives, [β]-glucan, arabinoyl xylan, carrageenan, pectin, glycogen, fucoidan, chondroitin, dermatan, heparin, heparin, pentosan, keratin, alginate / alginate, cyclodextrin, and their salts and derivatives, including their esters and sulfates.

[0175] Preferred pharmaceutically acceptable carriers include aqueous carriers or diluents. Examples of suitable aqueous carriers for use in the pharmaceutical compositions of the present invention include water, buffered water, and saline. Other examples of carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. For example, appropriate flowability can be maintained by using coating materials such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In many cases, it is preferred to include isotonic agents in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride.

[0176] Pharmaceutical compositions may include pharmaceutically acceptable antioxidants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifiers, and dispersants. Prevention of microbial growth can be ensured through the sterilization procedures described above and by adding various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. The addition of isotonic agents, such as sugars and sodium chloride, to the composition may also be desirable. Furthermore, prolonged absorption of injectable drug formulations can be achieved by adding absorption-delaying agents, such as aluminum monostearate and gelatin.

[0177] Therapeutic compositions must generally be sterile and stable under the conditions of preparation and storage. Pharmaceutical compositions can be formulated into solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.

[0178] A sterile injectable solution can be prepared by adding the required amount of an active agent (e.g., an antibody) to a suitable solvent, and, if necessary, adding one or more of the components listed above, followed by microfiltration sterilization. Generally, dispersions can be prepared by adding an active agent to a sterile medium containing a base dispersion medium and other desired components from the list of components above. For sterile powders used to prepare sterile injectable solutions, preferred methods of preparation include vacuum drying and freeze-drying (lyophilization), which allow powders containing the active agent and any other desired components to be obtained from solutions previously sterile filtered.

[0179] Pharmaceutical compositions may contain additional active ingredients and therapeutic agents. The compositions of the present invention may contain one or more antigen-binding proteins, polynucleotides, or viral particles. They may also contain additional therapeutic or prophylactic active agents.

[0180] Depending on the route of application, the antigen-binding protein of the present invention can be coated in a material to protect the antigen-binding protein from acids and other natural conditions that may cause the antigen-binding protein to become inactive or denatured.

[0181] The present invention further provides the antigen-binding protein, polynucleotide, carrier / virus particle, or composition of the present invention for use in treatment methods. Optionally, the treatment method includes administering an effective amount of the antigen-binding protein, polynucleotide, or carrier / virus particle of the present invention to a patient.

[0182] The present invention further provides a treatment method comprising administering to a patient an effective amount of the antigen-binding protein, polynucleotide, or carrier / virus particle of the present invention.

[0183] The present invention further provides the use of the antigen-binding protein, polynucleotide, carrier / virus particle, or composition of the present invention in the preparation of a medicament for a treatment method. Optionally, the treatment method comprises administering an effective amount of the polynucleotide or carrier / virus particle of the present invention to a patient.

[0184] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome at the necessary dose and within the required time frame, such as increasing the level of functional FVIII mimic antibodies in the subject (so that the production of functional antibodies is sufficient to alleviate the symptoms of hemophilia A).

[0185] In therapeutic applications, an antigen-binding protein, polynucleotide, or combination is administered to a subject already suffering from a condition or illness in an amount sufficient to cure, alleviate, or partially prevent the condition or one or more of its symptoms. Such therapeutic treatment may result in a reduction in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. An amount sufficient to achieve this purpose is defined as a “therapeutic effective amount.” The effective amount for a given purpose will depend on the severity of the disease or injury, as well as the subject’s weight and overall condition. In prophylactic applications, an antigen-binding protein, polynucleotide, or combination is administered to a subject who has not yet exhibited symptoms of a certain condition or illness in an amount sufficient to prevent or delay the onset of symptoms. Such an amount is defined as a “prophylactic effective amount.” As used herein, the term “subject” includes any vertebrate, typically any mammal, such as a human or horse. Humans are preferred as subjects.

[0186] The antigen-binding protein of the present invention can be administered via one or more routes of administration, employing one or more methods known in the art. As those skilled in the art will understand, the route and / or manner of administration will vary depending on the desired outcome. Preferred routes of administration for the antigen-binding protein, antibody, or composition of the present invention include intravenous, subcutaneous, intraocular, intradermal, intraperitoneal, intraspinal, or other parenteral administration routes, such as by injection or infusion. As used herein, the phrase “parenteral administration” refers to a method of administration other than enteral and local administration, typically by injection. Administration can be rectal, oral, ocular, local, transepidermal, or via mucosal routes. Administration can be local, including peritumoral, adjacent, intratumoral, to the tumor margin, intralesional, perilesional, intracavitary infusion, intravesical / intravesical administration, or inhalation. In a preferred embodiment, the pharmaceutical composition is administered intravenously or subcutaneously.

[0187] The appropriate dosage of the antigen-binding protein of the present invention can be determined by a skilled medical practitioner. The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may vary in order to obtain a quantity of active ingredient that, for a particular patient, composition, and route of administration, effectively achieves the desired therapeutic response without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the particular therapeutic agent used, the route of administration, the time of administration, the excretion rate of the therapeutic agent, the duration of treatment, other drugs, compounds, and / or substances used in combination with the particular composition used, the age, sex, weight, condition, general health status, and medical history of the patient being treated, and similar factors well known in the medical field.

[0188] For example, an appropriate dose of antigen-binding protein can be in the range of approximately 0.1 µg / kg to approximately 100 mg / kg of the patient's body weight. For instance, an appropriate dose could be approximately 1 µg / kg of body weight to approximately 50 mg / kg of body weight per week, approximately 100 µg / kg of body weight to approximately 25 mg / kg of body weight per week, or approximately 10 µg / kg of body weight to approximately 12.5 mg / kg of body weight per week.

[0189] Appropriate doses may be approximately 1 µg / kg body weight to approximately 50 mg / kg body weight per day, approximately 100 µg / kg body weight to approximately 25 mg / kg body weight per day, or approximately 10 µg / kg body weight to approximately 12.5 mg / kg body weight per day.

[0190] Dosing regimens can be adjusted to provide the optimal desired response (e.g., therapeutic response). For example, a single bolus injection can be administered, or several fractionated doses can be administered over a period of time, or the dose can be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and dosage uniformity, it is particularly advantageous to formulate the parenteral composition in unit dosage form. As used herein, unit dosage form refers to a physically discrete unit suitable for use in the subject to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect when used with the required drug delivery system.

[0191] Antigen-binding proteins can be administered in single or multiple doses. Multiple doses can be administered via the same or different routes and at the same or different sites. Alternatively, antigen-binding proteins can be administered as a sustained-release / extended-release formulation, in which case fewer administrations are required. Dosage and frequency can vary depending on the half-life of the antigen-binding protein in the patient and the desired duration of treatment. The dosage and frequency of administration can also vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic use, relatively low doses can be administered at relatively infrequent intervals over a longer period. In therapeutic use, relatively high doses can be administered, for example, until the patient exhibits partial or complete remission of disease symptoms.

[0192] The combined administration of two or more agents can be achieved in a variety of different ways. In one embodiment, the antigen-binding protein of the present invention can be administered together with other agents in a single composition. In another embodiment, the antigen-binding protein of the present invention can be administered in separate compositions as part of a combination therapy with other agents. For example, the antigen-binding protein of the present invention can be administered before, after, or simultaneously with other agents.

[0193] Optionally, the treatment method is gene therapy. Gene therapy"This relates to the application of the vector / virus particles of the present invention, which are capable of expressing transgenes (such as polynucleotides encoding FVIII mimic antigen-binding proteins) in the host to which they are applied."

[0194] Optionally, the dose of the vector / virus particles administered is less than 1 x 10^6 per kg of patient body weight. 11 Less than 1 x 10 12 Less than 5 x 10 12 Less than 2 x 10 12 Less than 1.5 x 10 12 Less than 3 x 10 12 Less than 1 x 10 13 Less than 2 x 10 13 or less than 3 x 10 13 One vector genome. Optionally, the dosage of the polynucleotide, viral particle, or composition is at least 4.5 x 10^6 mg / kg of patient body weight. 11 Or 4.5 x 10 11 Up to 1 x 10 12 One vector genome (vg / kg). Optionally, the dosage of polynucleotides, viral particles, or compositions administered is less than 5 x 10⁻⁶. 11 vg / kg. Optionally, the dosage of polynucleotides, viral particles, or compositions administered is 4.5 x 10⁻⁶. 11 vg / kg up to 5 x 10 11 vg / kg or 4.5 x 10 11 vg / kg up to 4.9 x 10 11 vg / kg. Optionally, the applied polynucleotide or viral particles are produced by mammalian cells and / or have characteristics of vectors produced by mammalian viral vector production cells and are distinct from those produced in insect viral vector production cells (e.g., baculovirus systems).

[0195] Optionally, the administration of a given dose of vector / virus particles – quantified by the amount of vector genome – is achieved by titrating the vector genome using qPCR. In principle, qPCR primers can be designed to bind any portion of the recombinant vector genome not shared with the wild-type genome, but the use of primer template sequences very close to the ITR is not recommended, as this may result in artificially inflated vector genome titer measurements. Optionally, the vector genome is quantified by quantitative polymerase chain reaction (qPCR), which utilizes primers targeting promoter regions, such as the promoter region of a transgene cassette (sometimes also called a transgene expression cassette).

[0196] Methods for performing qPCR are known to those skilled in the art. Using a real-time PCR cycler and DNA-binding dyes such as SYBR Green (ThermoFisher Scientific), the amplification of newly formed double-stranded amplicon can be detected in real time. Then, a known amount of qPCR template genetic material (e.g., promoter region) can be serially diluted to create a standard curve, and the sample vector genomic titer can be obtained by interpolation based on this standard curve.

[0197] In one aspect, the polynucleotides, viral particles, or compositions of the present invention are used to achieve stable levels of antigen-binding protein activity in subjects. The term "..." Stable antigen-binding protein activity levels "" refers to the antigen-binding protein activity level being maintained at or above a certain level for a continuous period of at least 5 weeks. In some embodiments, the antigen-binding protein activity level is maintained at or above a certain level for a continuous period of at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 weeks. The activity level of the antigen-binding protein can be determined by methods known to those skilled in the art, including monitoring FVIII mimicry activity.

[0198] The present invention includes a method for treating hemophilia A in a subject, the method comprising administering to the subject a therapeutically effective amount of a disclosed antigen-binding protein and / or a polynucleotide encoding said antigen-binding protein and / or a viral particle containing said polynucleotide, thereby treating hemophilia.

[0199] The present invention also relates to the use of the disclosed antigen-binding protein and / or the polynucleotide encoding the antigen-binding protein and / or viral particles containing the polynucleotide in the treatment of hemophilia A. Furthermore, the present invention relates to the use of the disclosed antigen-binding protein and / or the polynucleotide encoding the antigen-binding protein and / or viral particles containing the polynucleotide in the preparation of a medicament for the treatment of hemophilia A.

[0200] sequence list

[0201] SEQ ID NO: 1: FIX-binding antigen-binding domain LCDR 1

[0202] RNIERQ

[0203] SEQ ID NO: 2: FIX-binding antigen-binding domain LCDR 2

[0204] QAS

[0205] SEQ ID NO: 3: FIX-binding antigen-binding domain LCDR 3

[0206] QQYSDPPLT

[0207] SEQ ID NO: 4: FIX-binding antigen-binding domain HCDR 1

[0208] GFTFSYYD

[0209] SEQ ID NO: 5: FIX-binding antigen-binding domain HCDR 2

[0210] ISPSGQST

[0211] SEQ ID NO: 6: FIX-binding antigen-binding domain HCDR 3

[0212] ARRTGREYGGGWYFDY

[0213] SEQ ID NO: 7: FX-binding antigen-binding domain LCDR 1

[0214] QSLVYSDGNTY

[0215] SEQ ID NO: 8: FX-binding antigen-binding domain LCDR 2

[0216] KVS

[0217] SEQ ID NO: 9: FX-binding antigen-binding domain LCDR 3

[0218] MQGTHWPPT

[0219] SEQ ID NO: 10: FX-binding antigen-binding domain HCDR 1

[0220] GFTFSSYA

[0221] SEQ ID NO: 11: FX-binding antigen-binding domain HCDR 2

[0222] ISYDGSHK

[0223] SEQ ID NO: 12: FX-binding antigen-binding domain HCDR 3

[0224] ARATTAARNGLDI

[0225] SEQ ID NO: 13: Light chain variable region of the FIX-binding antigen-binding domain (CDR shown in bold)

[0226] DIQMTQSPSSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIK

[0227] SEQ ID NO: 14: Heavy chain variable region of the FIX-binding antigen-binding domain (CDR shown in bold)

[0228] QVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSS

[0229] SEQ ID NO: 15: Light chain variable region of the FX-binding antigen-binding domain (CDR shown in bold)

[0230] DIVMTQSPLSLHVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKITRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK

[0231] SEQ ID NO: 16: Heavy chain variable region of the FX-binding antigen-binding domain (CDR shown in bold)

[0232] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSHKYYADSVKGRFTISRDSKDTLYLQMNSLGAEDTAVYYCARATTAARNGLDIWGQGTTVTVSS

[0233] SEQ ID NO: 17: FIX binding scFv

[0234] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSS

[0235] SEQ ID NO: 18: FX-binding scFv

[0236] EVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSHKYYADSVKGRFTISRDSSKDTLYLQMNSLGAEDTAVYYCARATTAARNGLDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLHVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKITRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIK

[0237] SEQ ID NO: 19: Amino acid sequence of Bi8

[0238] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSHKYYADSVKGRFTISRDSSKDTLYLQMNSLGAEDTAVYYCARATTAARNGLDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLHVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKITRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIKHHHHHH

[0239] SEQ ID NO: 20: 3xG4S linker region

[0240] GGGGSGGGGSGGGGS

[0241] SEQ ID NO: 21: Nucleotide sequence of Bi8

[0242]

[0243] SEQ ID NO: 22: Nucleotide sequence of FIX-binding scFv

[0244] ACATCGACATCCAGATGACACAGTCACCCAGCTCACTCTCTGCCTCAGTAGGGGACCGAGTGACAATCACATGTAAGGCCTCCAGAAACATTGAGAGGCAGCTCGCATGGTACCAACAGAAGCCGGGACAGGCTCCGGAACTGCTGATTTATCAGGCATCACGCAAGGAGAGTGGCGTCCCCGATCGGTTCTCTGGCAGTAGATACGGCACTGACTTTACACTGACTATTTCTAGCCTTCAGCCAGAAGATATCGCTACTTACTATTGCCAGCAGTACTCCGACCCCCCGTTGACATTTGGCGGCGGAACGAAAGTCGAAATCAAAGGCGGTGGCGGCTCTGGCGGAGGTGGCTCTGGCGGAGGCGGCAGCCAAGTGCAACTGGTCGAGTCTGGTGGCGGCCTCGTGCAGCCAGGAGGTTCACTTAGACTGTCCTGCGCTGCTAGTGGCTTTACTTTCAGCTACTACGACATCCAGTGGGTGCGCCAGGCCCCAGGGAAGGGGCTTGAGTGGGTAAGTTCTATTTCCCCATCCGGCCAGTCTACTTACTATCGGCGGGAGGTCAAAGGGAGATTCACCATCTCCCGCGACAATTCAAAGAACACATTGTACTTGCAAATGAACTCCCTGCGCGCAGAGGACACCGCCGTCTACTATTGCGCGAGACGCACAGGGCGGGAGTACGGTGGTGGTTGGTATTTCGATTATTGGGGGCAAGGAACACTCGTAACGGTCTCATCA

[0245] SEQ ID NO: 23: Nucleotide sequence of FIX-binding scFv - light chain

[0246] GACATCCAGATGACACAGTCACCCAGCTCACTCTCTGCCTCAGTAGGGGACCGAGTGACAATCACATGTAAGGCCTCCAGAAACATTGAGAGGCAGCTCGCATGGTACCAACAGAAGCCGGGACAGGCTCCGGAACTGCTGATTTATCAGGCATCACGCA AGGAGAGTGGCGTCCCCGATCGGTTCTCTGGCAGTAGATACGGCACTGACTTTACACTGACTATTTCTAGCCTTCAGCCAGAAGATATCGCTACTTACTATTGCCAGCAGTACTCCGACCCCCCGTTGACATTTGGCGGCGGAACGAAAGTCGAAATCAAA

[0247] SEQ ID NO: 24: Nucleotide sequence of FIX-binding scFv – light chain CDR1

[0248] AGAAACATTGAGAGGCAG

[0249] SEQ ID NO: 25: Nucleotide sequence of FIX-binding scFv – light chain CDR2

[0250] CAGGCATCA

[0251] SEQ ID NO: 26: Nucleotide sequence of FIX-binding scFv – light chain CDR3

[0252] CAGCAGTACTCCGACCCCCCGTTGACA

[0253] SEQ ID NO: 27: Nucleotide sequence of the FIX-binding scFv-heavy chain

[0254] CAAGTGCAACTGGTCGAGTCTGGTGGCGGCCTCGTGCAGCCAGGAGGTTCACTTAGACTGTCCTGCGCTGCTAGTGGCTTTACTTTCAGCTACTACGACATCCAGTGGGTGCGCCAGGCCCCAGGGAAGGGGCTTGAGTGGGTAAGTTCTATTTCCCCATCCGGCCAGTCTACTTACTATCGGC GGGAGGTCAAAGGGAGATTCACCATCTCCCGCGACAATTCAAAGAACACATTGTACTTGCAAATGAACTCCCTGCGCGCAGAGGACACCGCCGTCTACTATTGCGCGAGACGCACAGGGCGGGAGTACGTGGTGGTTGGTATTTCGATTATTGGGGGCAAGGAACACTCGTAACGGTCTCATCA

[0255] SEQ ID NO: 28: Nucleotide sequence of FIX-binding scFv-heavy chain CDR1

[0256] GGCTTTACTTTCAGCTACTACGAC

[0257] SEQ ID NO: 29: Nucleotide sequence of FIX-binding scFv-heavy chain CDR2

[0258] ATTTCCCCATCCGGCCAGTCTACT

[0259] SEQ ID NO: 30: Nucleotide sequence of FIX-binding scFv-heavy chain CDR3

[0260] GCGAGACGCACAGGGCGGGAGTACGGTGGTGGTTGGTATTTCGATTAT

[0261] SEQ ID NO: 31: Nucleotide sequence of FX-binding scFv

[0262] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATACACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTCATAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGTTCCAAGGACACGCTGTATCTGCAAATGAACAGTCTGGGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGCAACTACTGCTGCTCGAAATGGTCTTGATATCTGGGGGCAAGGGACCACGGTCACCGTCTCGAGTGGAGGCGGAGGGTCTGGGGGCGGCGGTAGCGGCGGAGGAGGAAGCGATATTGTGATGACCCAGTCTCCACTCTCCCTGCACGTCACCCTTGGACAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTTTCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAGATCACCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0263] SEQ ID NO: 32: Nucleotide sequence of the FX-binding scFv - light chain

[0264] GATATTGTGATGACCCAGTCTCCACTCTCCCTGCACGTCACCCTTGCAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTATACAGTGATGGAAACACCTACTGAATTGGTTTCAGCAGAGGCCAGGCCAATCTCCAAGGCGCCTAATTTATAAGGTT TCTAACCGGGACTCTGGGGTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACTGATTTCACACTGAAGATCACCAGGGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGGTACACACTGGCCTCCGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA

[0265] SEQ ID NO: 33: Nucleotide sequence of FX-binding scFv-light chain CDR1

[0266] CAAAGCCTCGTATACAGTGATGGAACACCTAC

[0267] SEQ ID NO: 34: Nucleotide sequence of FX-binding scFv – light chain CDR2

[0268] AAGGTTTCT

[0269] SEQ ID NO: 35: Nucleotide sequence of FX-binding scFv – light chain CDR3

[0270] ATGCAAGGTACACACTGGCCTCCGACG

[0271] SEQ ID NO: 36: Nucleotide sequence of the FX-binding scFv-heavy chain

[0272] GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTATGCTATACACTGGGTCCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATCATATGATGGAAGTCATAAATACTAC GCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGTTCCAAGGACACGCTGTATCTGCAAATGAACAGTCTGGGAGCCGAGGACACGGCTGTGTATTACTGTGCAAGAGCAACTACTGCTGCTCGAAATGGTCTTGATATCTGGGGGCAAGGGACCACGGTCACCGTCTCGAGT

[0273] SEQ ID NO: 37: Nucleotide sequence of FX-binding scFv-heavy chain CDR1

[0274] GGATTCACCTTCAGTAGCTATGCT

[0275] SEQ ID NO: 38: Nucleotide sequence of FX-binding scFv-heavy chain CDR2

[0276] ATATCATATGATGGAAGTCATAAA

[0277] SEQ ID NO: 39: Nucleotide sequence of FX-binding scFv-heavy chain CDR3

[0278] GCAAGAGCAACTACTGCTGCTCGAAATGGTCTTGATATC

[0279] SEQ ID NO: 40: Amino acid sequence of Bi8 with HLE

[0280] DIQMTQSPSSLSASVGDRVTITCKASRNIERQLAWYQQKPGQAPELLIYQASRKESGVPDRFSGSRYGTDFTLTISSLQPEDIATYYCQQYSDPPLTFGGGTKVEIKGGGGSGGGGSGGGGSQVQLVESGGGLVQPGGSLRLSCAASGFTFSYYDIQWVRQAPGKGLEWVSSISPSGQSTYYRREVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRTGREYGGGWYFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGVVQPGRSLRLSCAASGFTFSSYAIHWVRQAPGKGLEWVAVISYDGSHKYYADSVKGRFTISRDSSKDTLYLQMNSLGAEDTAVYYCARATTAARNGLDIWGQGTTVTVSSGGGGSGGGGSGGGGSDIVMTQSPLSLHVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKITRVEAEDVGVYYCMQGTHWPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFVASILWSGNNRDYADSVKGRFAISRDNAKNTAYLQMTSLKPEDTAVYYCAAGDGLGFYRSVNQYDYWGQGTQVTVSSHHHHHH

[0281] SEQ ID NO: 41: Amino acid sequence of albumin - targeting VHH

[0282] EVQLVESGGGLVQAGGSLRLSCTASGRTFTPYTMGWFRQAPGKEREFVASILWSGNNRDYADSVKGRFAISRDNAKNTAYLQMTSLKPEDTAVYYCAAGDGLGFYRSVNQYDYWGQGTQVTVSS

[0283] SEQ ID NO: 42: CDR1 sequence of albumin - targeting VHH

[0284] GRTFTPYTMG

[0285] SEQ ID NO: 43: CDR2 sequence of VHH targeting albumin

[0286] SILWSGNNRDYADSVKG

[0287] SEQ ID NO: 44: CDR3 sequence of VHH targeting albumin

[0288] GDGLGFYRSVNQYDY

[0289] SEQ ID NO: 45: Nucleotide sequence of Bi8 with HLE

[0290]

[0291] SEQ ID NO: 46: Nucleotide sequence of VHH targeting albumin

[0292] GAAGTGCAGCTCGTAGAATCAGGTGGTGGTCTTGTTCAAGCTGGGGGTTCACTGAGGTTGTCTTGTACTGCGAGTGGCCGAACCTTTACTCCGTACACGATGGGATGGTTTAGGCAAGCACCAGGAAAGGAGCGGGAATTTGTTGCATCTATTCTCTGGAGTGGAAACAATAGGGACTATGCAGAT AGTGTGAAGGGTCGCTTCGCCATTTCCCGGGACAATGCCAAGAACACAGCTTACCTCCAGATGACAAGCCTCAAGCCGGAGGATACAGCCGTATATTATTGCGCTGCCGGCGATGGTCTCGGGTTTTACAGAAGCGTGAATCAGTACGACTACTGGGGACAAGGCACTCAGGTTACTGTATCAAGC

[0293] SEQ ID NO: 47: Nucleotide sequence of the CDR1 sequence of VHH targeting albumin.

[0294] GGCCGAACCTTTACTCCGTACACGATGGGA

[0295] SEQ ID NO: 48: Nucleotide sequence of the CDR2 sequence of VHH targeting albumin.

[0296] TCTATTCTCTGGAGTGGAAACAATAGGGACTATGCAGATAGTGTGAAGGGT

[0297] SEQ ID NO: 49: Nucleotide sequence of the CDR3 sequence of VHH targeting albumin.

[0298] GGCGATGGTCTCGGGTTTTACAGAAGCGTGAATCAGTACGACTAC

[0299] Example

[0300] Example 1 - Materials and Methods

[0301] Preparation and purification of recombinant Bi8

[0302] The coding sequences for Bi8 or Bi8-HLE were cloned into the pcDNA3.1hygro(+) expression vector for protein expression, and recombinant antibodies were generated internally using transient transfection in a mammalian Expi293 expression system (Gibco). High-density Expi293F™ cells were routinely passaged in Expi293 expression medium at 37°C, 8% CO2, and 130 rpm. For transfection, cells were cultured at 1 x 10⁻⁶ cells / day prior to transfection. 6 Cells were seeded at a density of 1 viable cells / ml in Expi293 expression medium. The following day, the required volume of cell suspension was transfected with the pcDNA3.1hygro_Bi8 plasmid using ExpiFectamine™ according to the manufacturer's instructions. 16–18 hours post-transfection, a production enhancer was added to the cells as recommended to increase protein yield, and the culture was incubated for another 5 days. The production supernatant was collected and clarified by centrifugation (4000 g for 20 min) to remove cells and filtration through a 0.22 µm filter. The recombinant Bi8 antibody was purified by affinity chromatography using a 5 mL HiTrap™ column packed with MabSelect™ PrismA resin (Cytiva) on an ÄKTA avant 25 system (Cytiva). The purified antibody was eluted from the column at a lower pH using a buffer containing 50 mM glycine and 150 mM NaCl at pH 3, and immediately neutralized with 2% v / v 2 M Tris base solution at pH 8.3. After purification, the recombinant Bi8 was placed in HEPES buffer at pH 7.6 containing 50 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid and 150 mM NaCl for buffer replacement and stored at -80°C.

[0303] AAV production and purification

[0304] A liver-specific expression cassette containing Bi8 or Bi8-HLE coding sequences was inserted between the inverted terminal repeat (ITR) motifs of the pAAV-specific backbone (serum type 2) to generate a single-stranded transfer plasmid, and the AAV vector was generated using the human embryonic kidney HEK-293T cell line. One day prior to transfection, 1.5 x 10-1 7Cells were seeded in 150 mm culture dishes containing high-glucose DMEM (4.5 g / L Gibco) supplemented with 10% v / v fetal bovine serum (FBS). The pAAV transfer plasmid was transiently co-transfected with an adenovirus helper plasmid (HGTI) and an AAV8 capsid element plasmid using polyethyleneimine (PEI) to generate a serum-pseudogenized AAV vector. Twenty-four hours post-transfection, the transfection medium was replaced with fresh production medium (high-glucose DMEM), and a 2-day production process was initiated. At the end of production, cells and supernatant were collected.

[0305] Cells were lysed via freeze / thaw cycles to extract intracellular rAAV. Cell debris was removed by centrifugation (4000 g for 20 min), and the cell lysates and supernatant were clarified by filtration through a 0.22 μm filter and then mixed. Recombinant AAV was purified from the preparation by affinity chromatography using POROS-AAVX resin (Cytiva) on an ÄKTA avant 25 system. AAV was eluted in glycine buffer (50 mM) at pH 2.7 and then rapidly neutralized with 2 M Tris buffer (pH 8.8). Peak fractions were combined and dialyzed in phosphate-buffered saline (PBS) using a 10 kDa molecular weight cutoff Slide-A-Lyzer dialysis cartridge (Thermofisher). The dialyzed preparation was concentrated 10-fold using a 100 kDa Amicon centrifuge filter (Merck-Millipore). AAV titers were quantified by qPCR using primers annealed to the poly-A tail region in the expression cassette.

[0306] SDS-page migration

[0307] Recombinant Bi8 samples were prepared using NuPAGE LDS sample buffer (Invitrogen) with or without NuPAGE sample reducing agent (Invitrogen) and denatured at 90 °C for 10 min. 1 μg of denatured sample was loaded into a NuPAGE 4-12% bis-Tris pre-prepared gel (Invitrogen) and migrated in MOPS-SDS running buffer (Invitrogen) at 120–180 V. 5 μL of pre-stained PageRuler protein molecular weight standards (Thermofisher) were added to separate wells for molecular weight comparison. Migrating proteins were detected by overnight staining with PageBlue protein staining solution (Thermofisher).

[0308] alkaline gel

[0309] The integrity of the AAV-packaged transgenic DNA was assessed by migration onto a 0.8% agarose gel prepared with alkaline running buffer (50 mM NaOH, 1 mM EDTA). 30 μL of the AAV preparation was mixed with 6 μL of loading buffer (300 mM NaOH, 6 mM EDTA, 30% glycerol, 1.8% SDS, xylenecyanide) and loaded onto the gel. Electrophoresis was performed at 10 V at 4°C for 17.5 hours. The gel was then neutralized for 2 hours with gentle shaking in 3 gel volumes of 0.1 M Tris buffer (pH 8.0), washed with tap water, and subjected to 1 gel volume of 2x SYBR water diluted with 0.1 M NaCl. TM Gold (ThermoFisher), stained for 5 minutes in the dark. (ChemiDoc) TM Images are acquired on the imaging system (Biorad).

[0310] Colorimetric determination of FVIII simulated activity

[0311] The FVIII mimicry activity of Bi8 was assessed by measuring FIX-mediated FX to FXa activation using an FXa-specific S-2765 chromogenic substrate. In 96-well polystyrene flat-bottom half-well plates (Greiner Bio-one), 50 μL of coagulation factor solution containing 40 μM phospholipid-TGT (Rossix AB), 280 nM plasma-derived human FX (Coagadex®, Bio Product Laboratory), and 6 nM plasma-derived human FIXa (Haematologic Technologies) dissolved in running buffer (50 mM Tris base, 150 nM NaCl, 0.1% protease-free BSA, pH 7.8) was used. 25 μL of S-2765 chromogenic substrate (Chromogenix) was diluted to 1.2 mM with run buffer containing 20 mM CaCl2 and added to each well. Chromogenic transformation kinetics were immediately recorded on a SpectraMax m3 Molecular Devices instrument at 37°C and 405 nm for 60 minutes (1 read / min, 2-second oscillation before each read). A commercially available FVIII mimic antibody, emicizumab, was used as a positive control, derived from remaining vials of commercially available Hemlibra™ from the Royal Free Hospital, London.

[0312] Activated partial thromboplastin time (aPTT)

[0313] Citric acid-treated human plasma pools were prepared from at least 20 healthy volunteers, and 200 Bethesda units (BU) of an FVIII-neutralizing polyclonal antibody (PAHFVIII-S, Haematologica Technologies) were added to eliminate FVIII activity. Then, 350 nM of recombinant Bi8 was added to the induced hemophilia A plasma, and the mixture was incubated at room temperature for 15 minutes. Clotting time was measured on an ACL Top 700 coagulation analyzer (Werfen Limited) using a silica-based aPTT synthaSil reagent.

[0314] FXIa-triggered thrombin generation assay (TGA)

[0315] Thrombin generation was measured using a calibrated automated thrombin curve (CAT) method on a thrombin generation analyzer (Diagnostica Stago). As previously described, FVIII activity was neutralized in a pool of human plasma containing 200 BU of anti-FVIII antibody. The neutralized plasma was then supplemented with 350 nM recombinant Bi8 and incubated at room temperature for 15 minutes. For each condition, 80 μL of plasma was dispensed into an Immulon 2HB U-type plate (Diagnostica Stago) and mixed with 20 μL of trigger solution, which consisted of 2.35 nM human FXIa and 100 μM phospholipid-TGT (Rossix AB) diluted in running buffer (50 mM Tris base, 150 nM NaCl, 0.1% proteinase-free BSA, pH 7.8). The plate was incubated at 37°C for 10 minutes, and thrombin generation was initiated by automatically injecting 20 μL of preheated FluCa reagent. The time to peak concentration and intrinsic thrombin potential (ETP) were calculated as the time to reach maximum thrombin generation and the area under the thrombin generation curve, respectively.

[0316] Quantitative analysis of Bi8 expression

[0317] Bi8 levels in cell supernatant and mouse plasma samples were quantified using enzyme-linked immunosorbent assay (ELISA). 96-well polystyrene flat-bottom half-well plates (Greiner Bio-one) were coated with 5 µg / mL human FX protein (Coagadex®, Bio Product Laboratory) diluted in carbonate buffer (Na₂CO₃ 12.2 mM, NaHCO₃ 35 mM, pH 9.6) and incubated overnight at 4°C. The plates were washed three times in TBS.T (50 mM Tris base, 150 mM NaCl, 0.1% Tween 20, pH 7.8) and saturated with TBS.T + 3% BSA at 37°C for 1 hour. After three washing cycles, the samples were diluted in TBS.T + 1% BSA and incubated at 37°C for 2 hours. Unbound samples were washed three times in TBS.T., while bound antibodies were detected using a 6x histidine tag motif via HRP-labeled monoclonal anti-His-tagged antibody (clone J099B12, Biolegend) diluted 1:2500 in TBS.T + 1% BSA and incubated at 37°C for 2 hours. After the final three washes, the plate was incubated with slow-kinetic 3,3′,5,5′-tetramethylbenzidine (TMB) substrate and allowed to develop for 5–15 minutes. The reaction was terminated by adding 2 M H2SO4 at a volume ratio, and the absorbance was read at 450 nm on a SpectraMax m3 (Molecular Devices) within 30 minutes. A standard curve was constructed using purified recombinant B8.

[0318] HuH7 model

[0319] The human hepatocellular carcinoma cell line HuH7 was used as a human hepatocellular carcinoma model to evaluate the expression of Bi8 after infection with the AAV_Bi8 vector.

[0320] HuH7 cells were routinely cultured in high-glucose DMEM medium (Sigma-Aldrich) supplemented with 10% FBS. Cells were spaced at 3 x 10⁶ cells per well. 4 Cells were seeded at a density of 100 cells / well in flat-bottomed 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. On the day of transduction, the AAV vector was diluted in X-vivo medium (Lonza) to achieve a multiplicity of infection (MOI) of 100 cells / well. 3 Up to 5x10 6One vector genome (vg) was collected. The cell supernatant was discarded and replaced with 100 μL of AAV preparation, and incubated for another 24 hours. Then, the AAV-containing medium was discarded and replaced with 200 μL of serum-free DMEM medium, and incubated for another 2 days. The supernatant was collected, clarified by centrifugation (4000 g for 15 minutes), and further used to assess the expression level of AAV-derived Bi8 and FVIII mimic activity.

[0321] Characterization of AAV_Bi8 vector in hemophilia A mice

[0322] In hemophilia A mice, the AAV vector encoding the Bi8 sequence was characterized. FVIII-deficient male mice with a C57 / Bl6 genetic background, aged 8–12 weeks, received a single intravenous injection of the AAV_Bi8 vector diluted in X-Vivo medium (Lonza) via the lateral tail vein. Animals were monitored for up to 8 weeks, with blood samples collected every 2 weeks via retroorbital puncture. These blood samples were placed in 10% 0.139 M sodium citrate anticoagulant to assess transgenic construct expression.

[0323] To assess the procoagulant potential of AAV_Bi8 in vivo, treated mice were challenged in a tail vein transection (TVT) hemorrhage model 3 weeks after AAV infusion, as previously described in (Johansen PB et al., Haemophilia 2016). Mice were first anesthetized with isoflurane gas (Dechra) according to a non-recovery procedure. Five minutes prior to measurement, a mixture of 100 U / kg human FIX (BeneFix, Pfizer) and FX (Haematologic Technologies) was administered via retroorbital injection. The lateral tail vein was transected, and the severed tail was subsequently immersed in preheated saline (37°C) and blood was collected for 45 minutes. The amount of hemoglobin in the collection tube was measured spectrophotometrically at 416 nm, and the blood loss volume was quantified by calculation based on a standard curve.

[0324] Anti-drug antibody ELISA assay

[0325] As previously described, the presence of anti-drug antibodies (ADAs) targeting Bi8 in mouse plasma was detected using an ELISA method. Briefly, 96-well flat-bottom half-wells were coated overnight with 5 μg / mL recombinant Bi8 in carbonate buffer. Plasma samples from AAV-treated mice were diluted 1:20 in TBS.T + 1% BSA and incubated at 37°C for 2 hours. Potential ADAs binding to Bi8 were then detected using HRP-labeled goat polyclonal anti-mouse Fc (Invitrogen, A16084) diluted 1:5000 in TBS.T + 1% BSA and incubated at 37°C for 2 hours. Positive controls were performed by adding different concentrations of monoclonal anti-His-tagged antibody (Biolegend, 652502) to untreated mouse plasma to mimic the presence of Bi8-targeting antibodies.

[0326] Example 2 - Design of a single-chain FVIII mimic antibody compatible with the expression cassette of the AAV vector

[0327] Full-length bispecific immunoglobulin G (IgG), including FVIII mimic antibodies already marketed for the treatment of hemophilia A or in clinical development, are complex multimeric proteins composed of at least three or four different peptide chains that require precise assembly to produce functional molecules. Their size and the complexity of this assembly process make these antibody forms poorly compatible with the design of short-sized expression cassettes, which are necessary for stable expression via AAV-mediated gene transfer.

[0328] To address this limitation, Bi8 was engineered into a small-sized single-chain FVIII mimic antibody, which simultaneously eliminated the difficulties associated with size or multimer assembly and bispecific antibodies. Single-chain variable fragments (scFv) targeting human coagulation FIX and FX were fused together via flexible linkers made of three repeating G4S motifs, forming a bispecific tandem scFv with a molecular weight of approximately 54.5 kDa. Figure 1 After being produced in mammalian cells and purified by affinity chromatography, the migration pattern of recombinant Bi8 antibody (recBi8) was assessed by electrophoresis under reducing and non-reducing conditions. Figure 2 Under both conditions, recBi8 exhibited a single separated band on Coomassie blue gel, with a size consistent with the expected size of bispecific tandem scFv, and only intact material was observed.

[0329] Example 3 - In vitro evaluation of the FVIII simulation potential of recombinant Bi8

[0330] The function of the FVIII mimic antibody is to enhance the FIXa-mediated catalytic conversion of FXa to activated FXa by simultaneously binding to two coagulation factors, and therefore may depend on the binding orientation between the bispecific antibody and its two targets. To assess whether the bispecific tandem scFv form used to design Bi8 could support the catalytic reaction, its FVIII mimic activity was measured in a dynamic colorimetric assay reproducing this enzymatic reaction. Figure 3 Although FXa activation is unobservable or only minimally observed in the absence of bispecific antibodies, stepwise conversion of the chromogenic substrate in the presence of recBi8 demonstrated that this bispecific form exhibits FVIII mimicry activity and successfully enhanced FIXa-mediated FX catalytic activation. Furthermore, the kinetic profile obtained using recBi8 was identical to that of the positive control prepared using the marketed FVIII mimicry antibody emecizumab, which has demonstrated clinical efficacy. Figure 3 ).

[0331] To further evaluate the strength of its FVIII mimicry activity, the ability of recBi8 to correct coagulation defects in human hemophilia A plasma was assessed in an aPTT assay. In this case, the lack of FVIII significantly increased the clotting time of hemophilia A plasma (mean ± SD; 99.8 ± 5.6 seconds) compared to the plasma pool of healthy individuals (30.6 ± 1.8). However, when 350 nM of recBi8 was added to hemophilia A plasma, the aPTT clotting time was significantly reduced to a mean of 19.0 ± 3.2 seconds, demonstrating the procoagulant activity of this single-chain FVIII mimic antibody. Figure 4 In addition, the ability of recBi8 to induce thrombin formation, a key parameter in the coagulation cascade, was evaluated in vitro. When added at the same concentration of 350 nM, the presence of recBi8 successfully restored thrombin formation in hemophilia A plasma upon activation of FXIa-triggered coagulation. Figure 5 A). Interestingly, compared to the normal plasma pool, the time to peak concentration corresponding to the time required to reach the maximum level of thrombin formation was significantly shortened, but not significantly (A). Figure 5 (B) This is a feature previously described in other FVIII mimic antibodies. However, in the presence of recBi8, the intrinsic thrombin potential (ETP) was comparable to that of the normal plasma pool (1574 ± 239 and 1592 ± 144 nM·min, respectively, mean ± SD), supporting the conclusion that recBi8 exhibits FVIII mimic activity and is fully capable of correcting the FVIII deficiency in hemophilia A plasma. Figure 5 B).

[0332] Example 4 - Engineering and in vitro evaluation of AAV vectors encoding the Bi8 sequence

[0333] The expression cassette for adapting Bi8 to the AAV vector pathway was designed to specifically target liver transduction. The Bi8 coding sequence was inserted downstream of the liver control region enhancer / human α-1 antitrypsin promoter (HCR hAAT) complex, which drives strong liver-specific expression of the transgene. Figure 6 A). After adding the polyadenylated region and inverted terminal repeat (ITR) required for AAV packaging, the Bi8 transgenic expression cassette was 4.4 kb in length, which is well within the packaging capacity range of the AAV capsid (which can hold up to 5 kb). This was confirmed by evaluating the pattern of the packaged transgenic DNA on an alkaline gel after generating and purifying AAV particles using the AAV8 capsid serotype. Figure 6 B). As expected, AAV_Bi8 showed a strong DNA band at 4.4 kb, corresponding to the complete transgene cassette.

[0334] The ability of the AAV_Bi8 vector to induce expression of a fully functional transgenic Bi8 construct was evaluated in vitro using the human hepatocyte cell line Huh7. When transduction was performed with AAV_Bi8 at an increased MOI (range of 10-1 cells per cell), the expression of the fully functional transgenic Bi8 construct was assessed. 3 Up to 5x10 6 One vector genome (vg), HuH7 cells showed dose-dependent expression of transgenic Bi8 detectable in the supernatant. Figure 7 A). The FVIII-mimicking activity of transgenic Bi8 produced from transduced HUH7 was further evaluated in a chromogenic assay. The highest MOI was 5 x 10⁻⁶. 6 The bispecific antibodies generated were normalized to the activity of the resulting recombinant Bi8. As expected, the transgenic Bi8 obtained by AAV-mediated transduction of HuH7 cells exhibited full-function FVIII mimic activity, with an activity / antigen ratio of 91.1 ± 13.5% of that of the recombinant Bi8 molecule. Figure 7 B).

[0335] Example 5 - Stable expression of Bi8 in hemophilia A mice after AAV-mediated gene transfer

[0336] In a mouse model of hemophilia A, transgenic expression of Bi8 was evaluated following AAV-mediated gene transfer. Male FVIII-deficient mice were administered a single AAV_Bi8 infusion via intravenous injection into the tail vein, using two different vectors at doses of 4e. 11 and 4e 12vg / kg. Then, the treated animals were monitored for 8 weeks to measure (vg / kg). Figure 8 The circulating concentration of transgenic Bi8 in plasma samples was measured every two weeks. Dose-dependent expression of transgenic Bi8 was observed as early as week 2, at a concentration of 4 mg / L. 11 and 4e 12 In mice treated with AAV_Bi8 at vg / kg, the circulating levels were measured to be 5.7 ± 3.0 and 39.1 ± 10.6 nM, respectively (mean ± SD). Figure 9 A). Interestingly, for both concentrations of the AAV vector, Bi8 transgene expression reached its maximum from week 2 and remained stable until week 8, with no significant change in the mean circulating level. Figure 9 A).

[0337] Then, plasma samples collected at week 8 were used to titrate any potential anti-drug antibodies (ADAs) targeting Bi8. While positive controls incubated with a commercially available mouse monoclonal antibody targeting Bi8 (a histidine tag) clearly showed the formation of Bi8-antibody complexes detectable by ELISA, samples from mice receiving 4e... 11 and 4e 12 Plasma samples from mice treated with two doses (vg / kg) remained negative. Given that the detection limit for ADA is 20 ng / mL, this loss of signal indicates that prolonged exposure of hemophilia A mice to circulating levels of Bi8 does not lead to the formation of detectable ADA. Figure 9 B).

[0338] Example 6 - Correction of bleeding tendency in hemophilia A mice after AAV-mediated gene transfer

[0339] The therapeutic potential of AAV_Bi8 in correcting bleeding was evaluated in hemophilia A mice. As previously described, male FVIII-deficient mice received a single infusion of AAV_Bi8 at a dose of 4e 11 4e 12 and 1.2e 13 vg / kg ( Figure 8 Bi8 transgene expression was measured 2 weeks after AAV infusion, and the circulating levels of FVIII mimic antibody for the three doses were 4.5 ± 2.3, 47.4 ± 16.0, and 178.8 ± 4.6 nM, respectively, expressed as mean ± SD. Figure 10 A).

[0340] In a tail vein transection (TVT) model optimized specifically for FVIII-mimicking bispecific antibodies, the bleeding characteristics of AAV-treated mice were evaluated, as previously described (Johansen PB et al., Haemophilia 2016; Ferriere, Peyron et al. 2020). In this model, human coagulation FIX and FX were injected as a bolus 5 minutes before the assay, and blood loss was recorded over a period of 45 minutes after tail vein transection. Figure 8 Following TVT induction, untreated hemophilia A mice exhibited a pronounced bleeding phenotype, with blood loss ranging from 516.7 to 929.1 µL (median = 732.0 µL). Figure 10 B)). However, all three groups of animals treated with AAV_Bi8 showed evidence of hemostasis correction of the bleeding phenotype. In fact, most AAV-treated mice showed reduced bleeding tendency, below the 75th percentile of the control group. Treatment response was dose-dependent, for 4e 11 4e 12 and 1.2e 13 In the vg / kg group, the median blood loss was significantly reduced to 372.0, 78.5, and 12.0 µL, respectively. Figure 10 B). 1.2e 13 The vg / kg group achieved blood loss levels comparable to wild-type animals, thus establishing the efficacy of the AAV vector in delivering a functional single-chain FVIII mimic antibody at a therapeutic level sufficient to correct the bleeding phenotype in hemophilia A mice.

[0341] Example 7 - Prolonged half-life of single-chain FVIII mimic antibody Bi8

[0342] Antibodies employing single-chain tandem scFv, such as Bi8, typically have a short half-life (1-2 hours) in humans. However, fusion with the albumin-binding moiety (which interacts with the neonatal Fc receptor (FcRn) pathway) can prolong the circulating half-life through an FcRn-mediated recycling mechanism. Therefore, expressing antibodies with longer half-lives after gene transfer may increase homeostasis, potentially reducing the AAV vector dose required to achieve therapeutic effects.

[0343] A Bi8 variant (Bi8-HLE) with a longer half-life was developed by fusing a camel-derived albumin-binding heavy-chain antibody fragment (VHH) to the C-terminal portion of a single-chain FVIII mimic antibody. Linking VHH downstream of an FX-targeting scFv and incorporating a triple-repeated G4S motif resulted in a trispecific single-chain antibody with a molecular weight of approximately 69.0 kDa. Figure 11The migration patterns of recombinant Bi8 and Bi8-HLE generated in mammalian cells were assessed by SDS-Page electrophoresis. For both constructs, a single separated band was observed at the expected size, and only intact antibodies were visible. Figure 12 A). The FVIII mimicry activity of Bi8-HLE was further evaluated using the colorimetric assay described previously. In the assay, increasing concentrations of Bi8-HLE induced dose-dependent activation of FX to FXa ( Figure 12 B). Furthermore, the FVIII mimicry activity of Bi8-HLE is comparable to that of the parental Bi8 antibody, demonstrating that the biological activity of the FVIII mimicry antibody is unaffected by the presence of albumin-binding VHH fused to the C-terminus. Figure 12 B).

[0344] Example 8 - In vitro evaluation of AAV vector encoding Bi8-HLE

[0345] The coding sequence for Bi8-HLE was cloned using the same liver-specific expression cassette previously used for parental Bi8 and is under the control of the HCR hAAT promoter complex. Figure 6 A). The presence of albumin-binding VHH fused to the C-terminus of a single-chain bispecific antibody extends the total size of the transgenic cassette to 4.7 kb, which is still within the packaging capacity range of the AAV capsid. Figure 13 A). In fact, after generating AAV vectors using the AAV8 capsid serotype and encoding Bi8 or Bi8-HLE, we confirmed the integrity of the packaged DNA by electrophoresis on an alkaline gel. Both constructs showed single DNA bands at 4.4 and 4.7 kb, respectively, corresponding to complete expression cassettes ( Figure 13 B).

[0346] The ability of the recombinant AAV vector to induce Bi8 or Bi8-HLE expression was further tested in the HuH7 cell model. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 6 In cells receiving a single dose of AAV vector per vg / cell, antibody concentrations detected in the supernatant 48 hours post-transduction were within a comparable range between the Bi8 and Bi8-HLE variants, with mean ± SD of 92.7 ± 63.8 and 173.7 ± 77.0 pM, respectively. Figure 14 This result demonstrates that AAV-mediated Bi8 expression in cellular systems is unaffected by the presence of albumin-binding elements and highlights the therapeutic potential of this construct in AAV-based gene therapy that induces the expression of single-chain FVIII mimic antibodies with extended half-life.

[0347] Implementation Plan

[0348] 1. A viral particle comprising a polynucleotide of length not exceeding 5.0 kb, the polynucleotide encoding a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDR)1, LCDR2, and LCDR3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3;

[0349] For the first antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 1; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 2; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 3; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 4; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 5; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 6, and...

[0350] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0351] 2. The viral particle of embodiment 1, wherein the polynucleotide encodes a single-stranded Fv protein (scFv) containing the first antigen-binding domain and a single-stranded Fv protein containing the second antigen-binding domain.

[0352] 3. The viral particle according to embodiment 1 or 2, wherein in the first antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13, and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14, and wherein in the second antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 15, and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 16.

[0353] 4. An antigen-binding protein composed of a single polypeptide chain, wherein the single polypeptide chain is composed of a first antigen-binding domain and a second binding domain, wherein the first antigen-binding domain selectively binds to coagulation FIX, and the second binding domain selectively binds to coagulation FX, and wherein each of the first antigen-binding domain and the second binding domain comprises a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDR)1, LCDR2 and LCDR3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR)1, HCDR2 and HCDR3;

[0354] The first antigen-binding domain selectively binds to coagulation FIX; and

[0355] For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO: 12.

[0356] 5. The antigen-binding protein according to embodiment 4, wherein the first antigen-binding domain comprises scFv, and the second antigen-binding domain comprises scFv.

Claims

1. An antigen-binding protein with FVIII mimicry activity, which consists of a single polypeptide chain.

2. The antigen-binding protein of claim 1, wherein the single polypeptide chain comprises a first antigen-binding domain and a second antigen-binding domain.

3. The antigen-binding protein of claim 2, wherein the first antigen-binding domain selectively binds to coagulation FIX, and the second binding domain selectively binds to coagulation FX.

4. The antigen-binding protein of claim 1 or 2, wherein the first antigen-binding domain and the second antigen-binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDR) 1, LCDR 2 and LCDR 3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR) 1, HCDR 2 and HCDR 3. The first antigen-binding domain selectively binds to coagulation FIX; and For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and wherein the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:

12.

5. The antigen-binding protein according to any one of claims 1-4, wherein the first antigen-binding domain and the second binding domain each comprise a light chain variable domain and a heavy chain variable domain, wherein the light chain variable domain comprises light chain complementarity-determining regions (LCDR) 1, LCDR 2 and LCDR 3, and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR) 1, HCDR 2 and HCDR 3. For the first antigen-binding domain, LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1; LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 2; and LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 3; and wherein the heavy chain variable domain comprises heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 4; HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 5; and HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 6; and For the second antigen-binding domain, LCDR1 contains the amino acid sequence shown in SEQ ID NO: 7; LCDR2 contains the amino acid sequence shown in SEQ ID NO: 8; and LCDR3 contains the amino acid sequence shown in SEQ ID NO: 9; and wherein the heavy chain variable domain contains heavy chain complementarity-determining regions (HCDR)1, HCDR2, and HCDR3, wherein HCDR1 contains the amino acid sequence shown in SEQ ID NO: 10; HCDR2 contains the amino acid sequence shown in SEQ ID NO: 11; and HCDR3 contains the amino acid sequence shown in SEQ ID NO:

12.

6. The antigen-binding protein of any one of claims 1-5, wherein for the first antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 13 and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 14, and for the second antigen-binding domain, the light chain variable domain comprises the amino acid sequence shown in SEQ ID NO: 15 and the heavy chain variable domain comprises the amino acid sequence shown in SEQ ID NO:

16.

7. The antigen-binding protein of any one of claims 1-6, wherein the first antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO: 17, and the second antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:

18.

8. The antigen-binding protein of any one of claims 1-7, wherein the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:

19.

9. The antigen-binding protein of any one of claims 1-8, wherein the antigen-binding protein further comprises a third antigen-binding domain, wherein the third binding domain binds to proteins that interact with FcRn.

10. The antigen-binding protein of claim 9, wherein the third antigen-binding domain binds to albumin.

11. The antigen-binding protein of claim 9 or 10, wherein the third antigen-binding domain comprises the sequences CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence shown in SEQ ID NO: 42; CDR2 comprises the amino acid sequence shown in SEQ ID NO: 43; and CDR3 comprises the amino acid sequence shown in SEQ ID NO:

44.

12. The antigen-binding protein of any one of claims 9-11, wherein the third antigen-binding domain comprises a VHH domain.

13. The antigen-binding protein of any one of claims 9-12, wherein the third antigen-binding domain comprises the amino acid sequence shown in SEQ ID NO:

41.

14. The antigen-binding protein of any one of claims 9-13, wherein the antigen-binding protein comprises the amino acid sequence shown in SEQ ID NO:

40.

15. A polynucleotide of length not exceeding 5.0 kb, comprising a nucleic acid sequence encoding an antigen-binding protein as defined in any one of claims 1 to 14.

16. The polynucleotide of claim 15, comprising a promoter located upstream of the nucleic acid sequence encoding the antigen-binding protein.

17. The polynucleotide of claim 16, wherein the promoter is a liver-biased or liver-specific promoter.

18. The polynucleotide of claim 17, wherein the liver-preferred or liver-specific promoter comprises a liver control region enhancer / human α-1 antitrypsin promoter (HCR hAAT) complex.

19. The polynucleotide of any one of claims 15 to 18, further comprising a first ITR located upstream of the promoter and a second ITR located downstream of the nucleic acid sequence encoding the antigen-binding protein.

20. The polynucleotide of any one of claims 15 to 19, further comprising a polyadenylated sequence located downstream of the nucleic acid sequence encoding the antigen-binding protein and upstream of the second ITR.

21. The polynucleotide of any one of claims 15 to 20, wherein the nucleic acid sequence encoding the antigen-binding protein encodes a single-stranded Fv protein (scFv) comprising the first antigen-binding domain and a single-stranded Fv protein comprising the second antigen-binding domain, optionally wherein the nucleic acid sequence encoding the antigen-binding protein encodes a third antigen-binding domain, wherein the third antigen-binding domain binds a protein that interacts with FcRn.

22. The polynucleotide of any one of claims 15 to 21, wherein the nucleic acid sequence encoding the antigen-binding protein comprises SEQ ID NO: 21 or SEQ ID NO:

45.

23. A viral particle comprising a polynucleotide as defined in any one of claims 15 to 22.

24. The virus particle of claim 23, wherein the AAV virus particle is preferably AAV8.

25. A composition comprising the antigen-binding protein, polynucleotide or viral particles as described in any of the preceding claims, and a pharmaceutically acceptable excipient.

26. An isolated host cell transformed with any one of the polynucleotides or viral particles according to claims 15 to 24.

27. The antigen-binding protein, polynucleotide, viral particle, or composition according to any one of claims 1 to 25, used in a treatment method.

28. The antigen-binding protein, polynucleotide, viral particle, or composition for use according to claim 27, wherein the treatment method comprises administering to a patient an effective amount of the antigen-binding protein, polynucleotide, viral particle, or composition according to any one of claims 1 to 25.

29. The antigen-binding protein, polynucleotide, viral particle, or composition for use according to claim 28, wherein the treatment method is a method for treating hemophilia.

30. The antigen-binding protein, polynucleotide, viral particle, or composition for use according to claim 29, wherein the treatment method is a method for treating hemophilia A.

31. An AAV genome comprising a polynucleotide encoding an FVIII mimic antigen-binding protein, used in gene therapy, optionally wherein the FVIII mimic antigen-binding protein is as defined in any one of claims 1 to 14.

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